Anti-il1rap antibodies

ABSTRACT

Provided herein are, inter alia, antibodies capable of binding Interleukin-1 receptor accessory protein (IL1RAP). The antibodies provided herein include novel light chain and heavy chain sequences and bind IL1RAP with high efficiency and specificity. The anti-IL1RAP antibodies provided herein are, inter alia, useful for the treatment of IL1RAP-expressing cancers such as AML.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/635,534, filed Jan. 30, 2020, which is a national stage entry under35 U.S.C. 371 of International Application No. PCT/US2018/044890, filedAug. 1, 2018, which claims priority to U.S. Provisional Application No.62/539,895, filed Aug. 1, 2017, all of which are hereby incorporated byreference in their entirety and for all purposes.

REFERENCE TO A SEQUENCE LISTING, A TABLE OR A COMPUTER PROGRAM LISTINGAPPENDIX SUBMITTED AS AN ASCII TEXT FILE

The Sequence Listing written in file 048440-667N01US_ST25.TXT, createdOct. 28, 2021, 38,297 bytes, machine format IBM-PC, MS Windows operatingsystem, is hereby incorporated by reference.

BACKGROUND OF THE INVENTION

Acute myeloid leukemia (AML) is a devastating hematopoietic malignancythat can lead to hematopoiesis failure and death. Despite increasingknowledge of the disease, current treatment options benefit only aminority of AML patients. The limited success of treatments is believedto be at least partially due to the inability of chemotherapy and/orother molecular targeting therapeutics to eliminate so-called leukemiastem cells (LSCs). Thus, there is a need in the art for treatments whichspecifically eliminate LSCs while sparing normal hematopoietic stemcells.

Immunotherapeutic approaches hold promise as an effective means oftreating patients suffering from AML. In order to be successful,however, immunotherapy must allow for the selective targeting anddestruction of LSCs. Provided herein are compositions and methods whichcure this and other needs in the art.

BRIEF SUMMARY OF THE INVENTION

In an aspect is provided an anti-interleukin-1 receptor accessoryprotein (IL1RAP) antibody including 3 a light chain variable domain anda heavy chain variable domain, wherein the light chain variable domainincludes: a CDR L1 as set forth in SEQ ID NO:1, a CDR L2 as set forth inSEQ ID NO:2 and a CDR L3 as set forth in SEQ ID NO:3; and wherein theheavy chain variable domain includes: a CDR H1 as set forth in SEQ IDNO:4, a CDR H2 as set forth in SEQ ID NO:5, and a CDR H3 as set forth inSEQ ID NO: 6.

In an aspect, an isolated nucleic acid encoding an antibody as providedherein, including embodiments thereof, is provided.

In an aspect is provided a pharmaceutical composition including atherapeutically effective amount of an antibody as provided herein,including embodiments thereof, and a pharmaceutically acceptableexcipient.

In an aspect is provided a method of treating cancer in a subject inneed thereof, the method including administering to a subject atherapeutically effective amount of an antibody as provided herein,including embodiments thereof, thereby treating cancer in the subject.

In an aspect is provided a recombinant protein including: (i) anantibody region including: (a) a light chain variable domain including aCDR L1 as set forth in SEQ ID NO:1, a CDR L2 as set forth in SEQ ID NO:2and a CDR L3 as set forth in SEQ ID NO:3; and (b) a heavy chain variabledomain a CDR H1 as set forth in SEQ ID NO:4, a CDR H2 as set forth inSEQ ID NO:5, and a CDR H3 as set forth in SEQ ID NO:6; and (ii) atransmembrane domain.

In another aspect is provided a recombinant protein including: (i) afirst antibody region capable of binding an effector cell ligand; and(ii) a second antibody region, including: (a) a light chain variabledomain comprising a CDR L1 as set forth in SEQ ID NO:1, a CDR L2 as setforth in SEQ ID NO:2 and a CDR L3 as set forth in SEQ ID NO:3; and (b) aheavy chain variable domain a CDR H1 as set forth in SEQ ID NO:4, a CDRH2 as set forth in SEQ ID NO:5, and a CDR H3 as set forth in SEQ ID NO:6.

In an aspect, an isolated nucleic acid encoding a recombinant protein asprovided herein, including embodiments thereof, is provided.

In an aspect is provided a pharmaceutical composition including atherapeutically effective amount of a recombinant protein as providedherein, including embodiments thereof, and a pharmaceutically acceptableexcipient.

In an aspect is provided a method of treating cancer in a subject inneed thereof, the method including administering to a subject atherapeutically effective amount of a recombinant protein as describedherein, including embodiments thereof, thereby treating cancer in thesubject.

In an aspect is provided a method of inhibiting proliferation of a cell,the method including: (i) contacting a cell with an anti-IL1RAP antibodyas provided herein including embodiments thereof, or a recombinantprotein as provided herein including embodiments thereof, therebyforming a contacted cell; and (ii) allowing the anti-IL1RAP antibody,the recombinant protein as provided herein including embodiments thereofto bind an IL1RAP on the contacted cell, thereby inhibitingproliferation of the cell.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-1D. Construction of human naive phage display library. FIG. 1A:Human antibody phage display library was constructed using peripheralblood mononuclear cells (PBMC) from 10 healthy donors. cDNA synthesizedfrom RNA of purified B cells was used as a template for antibody heavychain (VH) and light chain (VL) variable region amplification and fusedwith a linker (Gly4Ser)₃. The mixture of all scFvs were digested withrestriction enzyme and ligated with the linearize phagemid vector. Afterelectro-transformation, the TG1 bacteria were collected and glycerolstocks were made to store the library at −80° C. Library's quality wasdetermined by its diversity (FIGS. 1B-1C) and capacity. The librarycapacity was 9.72×10⁹ colonies. FIG. 1B: CDR3 length of both heavy chain(left) and light chain (right) from 50 randomly selected clones wereanalyzed. FIG. 1C: Coverage of different heavy chain (top) and lightchain (bottom) subfamily of the phage display library were analyzedbased on the sequencing results of 50 clones. The distribution of CDR3length and VH family was consistent with previously reported resultsfrom antibody repertoire next generation sequencing. FIG. 1D: Biopanningfor IL1RAP antibodies using recombinant human IL1RAP protein. Enrichmentwas observed at the fourth round. See also Table 2 which corresponds toFIG. 1D.

FIGS. 2A-2D. Anti-IL1RAP monoclonal antibodies selected from human naïvephage display library. The screening/sequencing process yielded 12unique antibody sequences. Thus Applicants selected the 12 positiveclones from their phage display library and the genes encoding heavy andlight chains of these antibodies from these clones were cloned to IgGexpression vectors for transient expression in expi293 cells. Antibodieswere purified by protein A resin for functional study. FIG. 2A: Bindingof 12 positive clones to recombinant IL1RAP protein. 1 μg/ml recombinantIL1RAP protein is coated to the 96-well plate at 4 degree overnight.Purified antibodies in three-fold serial dilution starting from 1p g/mlare added to each well. HRP-anti-human FC antibody is used as detectionantibody. FIG. 2B: EC₅₀ of 12 positive clones is calculated based on theELISA binding curve. FIG. 2C: Antibody Dependent Cellular Cytotoxicity(ADCC) detected by Cr⁵ release assay. Antibodies at 100 ng/ml are addedto 1×10⁴ Cr⁵¹ labeled MV4-11 cells. 2×10⁵ NK cells are used as effectorcells. The specific lysis percentage is detected at 10 hours afterincubation. FIG. 2D: Affinity of 1D5 detected by BiacoreT100. Combiningthe ELISA and ADCC results, Applicants selected 1D5 as the elite clonefor bispecific antibody and CAR-T development. Applicants then usedBiacore T100 to check its accurate KD. The KD of 1D5 was 21.2 nM.

FIGS. 3A-3C. Anti-CD3XIL1RAP bispecific antibody. FIG. 3A: Schematicrepresentation of anti-CD3XIL1RAP bispecific antibody design. Thebispecific antibody was assembled as scFv-FC. To facilitate heterodimerformation, the Knobs-into-Holes construct is applied to Applicants'bispecific antibody. The FC “knobs-into-holes” constructs were designedbased on the IgG-FC backbone as using a site-directed mutagenesis kit(Invitrogen, Carlsbad, Calif.). For the FC-knob construct, one mutationwas introduced in the CH3 domain (T366W, designated as knob) of oneIgG-FC backbone. For the FC-hole construct, three mutations wereintroduced into the CH3 domain (T366S, L368A, and Y407V, designated ashole) of another IgG-FC backbone. To circumvent light chain and heavychain mispairing, Applicants constructed a single chain variablefragment (scFv), linking the heavy and light chains of the same antibodyby a (Gly3Ser)₄ linker. The scFv fragments of anti-CD3 antibody andanti-IL1RAP protein antibody were then cloned into the IgG-FC-knob andIgG-FC-hole vector, respectively. To further abolish the non-specificbinding to AML cells by the FCγR, Applicants introduced double mutation(L234A, L235A) in the CH2 region. FIG. 3B: Expression of mono-specificand bispecific antibodies. SDS-PAGE gel result for purified proteinsshowed that anti-CD3-Knob-FC, anti-IL1RAP-Hole-FC, and anti-CD3xIL1RAPantibodies were all expressed. Lanes 1 to 3 are non-reduced protein:anti-CD3 knob antibody, anti-IL1RAP hole antibody, anti-CD3XIL1RAPbispecific antibody. Lanes 4 to 6 are reduced protein. Lane 7 isnon-reduced bispecific antibody and lane 8 is reduced bispecificantibody. As shown in FIG. 3B, the size of bispecific antibody (Lane 3)is between two mono-specific antibodies. FIG. 3C: Dual binding ofanti-CD3XIL1RAP antibody to T cell and IL1RAP protein was detected byflow cytometry. Bispecific antibody was incubated with T cells,recombinant IL1RAP protein with his tag was then added after it.FITC-anti-his antibody was used to detect the binding of IL1RAP proteinto the bispecific antibody.

FIGS. 4A-4I. Anti-CD3XIL1RAP bispecific antibody induces T-cellactivation and cytotoxicity in AML cell lines. FIG. 4A: Genomic MeanFluorescence Intensity (MFI) of IL1RAP expression in different AML celllines, Raji is used as negative control. Except for KG1a, whose genomicmedium fluorescence intensity (MFI) was slightly higher than Raji, MFIof the other six cell lines show significantly higher IL1RAP expressionthan Raji. FIG. 4B: Antibody copies bound per cell were compared indifferent AML cell lines. Based on the results shown in FIG. 4A and FIG.4B, Applicants decided to use HL60, MV4-11, THP1, and KG1a, as aspectrum of cell lines with high to low levels of IL1RAP, and Raji asnegative control for functional assays. FIG. 4C: T cell dependentcellular cytotoxicity (TDCC) was tested with the incubation of effectorT cells and target AML cells at an E:T (Effector:Target cell ratio)ratio of 3:1, the specific lysis percentage was checked at 48 hoursafter incubation. The specific lysis was determined by flow cytometry,calculated using the formula: % Specific lyses=(Targetcells^(+T cells)−Target cells^(+BsAb+T cells))/Targetcells^(+T cells))×100%. T cell activation is checked by quantificationof cytokines TNF-α (FIG. 4D), IFN-r (FIG. 4E), Granzyme B (FIG. 4F), andcell membrane markers CD69 (FIG. 4G) and CD25 (FIG. 4H). FIG. 4I:Different E:T ratios tested in the long term killing assay.

FIGS. 5A-5D. AML patient samples show high level IL1RAP expression. FIG.5A: Three representative IL1RAP expression level of AML patient sample.FIG. 5B: Genomic Mean Fluorescence Intensity (MFI) of IL1RAP expressionin different AML patient samples, PBMC was used as normal control.Compared to PBMC from healthy donor, all AML patient samples showedenhanced IL1RAP expression, shown by significantly higher MFI. FIG. 5C:T cell cytotoxicity is checked by a 48 hour long term killing assay atan E:T ratio of 5:1. FIG. 5D. T cell dependent cellular cytotoxicityassay (TDCC) with triplicates. CD34+ cells showed comparable IL1RAPexpression compared to the bulky leukemia cells. TDCC indicated 90% ofspecific killing in most samples, and 60% in sample 667, which has thelowest IL1RAP expression levels. No difference was observed betweensorted CD34+ and unsorted AML blasts.

FIGS. 6A-6B. Anti-CD3XIL1RAP effectively treats MV4-11 AML in NSG mice.FIG. 6A: Tumor growth was monitored by bioluminescence imaging. 1×10⁶MV4-11 cells were injected to each mouse via tail vein. Treatmentstarted on day 7 with 200 ug bispecific antibody and 3×10⁶ T cell permouse. The subsequent treatment and imaging time are indicated by thearrows below. Massive infiltration of leukemia was seen in the NSG micetransplanted with MV4-11^(Luci) cells treated with vehicle, T cellsonly, or control BsAb plus T cells. Instead, massive reduction ofleukemia burden was seen in the mice treated with T cells andanti-IL1RAPxCD3 BsAb at day 20, after one dose of treatment, andelimination at day 37, except for one mouse. One of the mice in thisgroup died of improper procedure on day 18 at the 4^(th) treatment. FIG.6B: Survival curve. Three out of 4 mice were still alive on day 40,versus none of the mice in the other three groups survived. Notably,mice treated with T cells or T cells and isotype control had also ashorter survival than the control mice, probably due to the toxicity ofT cells. Applicants also observed that the mice treated with BsAb werelosing weight, likely due to the cytokine release.

FIGS. 7A-7B. Jurkat cells transduced with IL1RAP CAR can be activatedwhen cocultured with AML cell lines. FIG. 7A: Schematic diagram of theCAR containing a modified immunoglobulin G4 hinge, a modifiedtransmembrane and intracellular signaling domain of CD28, and the CD3zsignaling domain. The T2A ribosomal skip sequence and the EGFRttransduction marker were also indicated. 293T cells were transfectedwith pRSV-Rev, pMDLg/pRRE, pMD2.G and pELNS CAR plasmids andsupernatants were collected at 24 hours and 48 hours and combined andconcentrated using high-speed ultracentrifugation. Lentivirus titer wasmeasured with HT-78 cells and stored at −80° C. until use. FIG. 7B:Jurkat cells were transduced with either IL1RAP CAR or mock CAR. CD69was detected for Jurkat cells cocultured with HL60, THP1, or KG1a cellswhich represent different IL1RAP expression levels separately. Rajicells were used as negative control. CD69 was detected for theactivation of IL1RAP-CAR cells. The activation of IL1RAP-CAR-Jurkatcells was observed when cocultured with IL1RAP expressing target cells,while the mock-CAR-Jurkat cells remained at rest. IL1RAP-CAR-Jurkatcells stayed inactive as mock-CAR-Jurkat cells when cocultured with Rajicells or alone.

FIGS. 8A-8I. IL1RAP as an immunotherapy target for AML. ILRAP expressionwas detected by flow cytometry on AML cell lines (FIG. 8A) and CD34enriched AML primary cells (FIG. 8B) but not on CD34+ enriched normalbone marrow cells (FIG. 8C). FIG. 8D: specific antigen-binding capacity(SABC) per cell as determined by flow cytometry in different AML celllines. FIG. 8E: fold change of IL1RAP mean fluorescence intensity wascompared between AML bulk cells (n=25), CD34+ enriched AML cells (n=18),and CD34+ enriched normal bone marrow cells (n=4). FIG. 8F: thepercentage of IL1RAP+, CD123+, CLL-1+ and CD33+ in CD34+ enriched AMLcells (n=9) were depicted with a symbol. FIG. 8G: fold change of meanfluorescence intensity on CD34+ enriched normal bone marrow cells (n=5),was compared for IL1RAP, CD123, CLL-1 and CD33. FIG. 8H: the percentageof IL1RAP+, CD123+, CLL-1+ and CD33+ in CD34+ enriched normal bonemarrow cells (n=5) were depicted with a symbol. FIG. 8I: the meanfluorescence intensity of IL1RAP on CD34+ enriched AML cells of G0 phaseand G1 phase was compared (2768 vs. 3181, no significant difference).

FIGS. 9A-9B. Anti-IL1RAP/CD3 bispecific antibody. FIG. 9A: ADCC efficacyof 12 candidate monoclonal antibodies determined by Cr⁵¹ release assay.1D5 showed the most potent ADCC efficacy. FIG. 9B: SDS-PAGE gel ofpurified antibodies. Reduced samples were loaded to lane 1-3,Non-reduced samples were loaded to lane 5-7. Anti-CD3 scFv-knob FC (1and 5), Anti-IL1RAP scFv-hole FC (2 and 6), and anti-IL1RAP/CD3bispecific antibody (3 and 7).

FIGS. 10A-10K. MM001 (CD3/IL1RAP bispecific antibody) induced T cellactivation and AML cell killing at different concentration and E:Tratio. FIG. 10A: T cell dependent cellular cytotoxicity (TDCC) wastested with the incubation of T cell and different AML cell lines (HL60,MV4-11, THP-1 and KG-1a) at a E:T ratio of 1:1, the specific lysispercentage was checked at 48 hours after incubation. The specific lysiswas determined by flow cytometry, calculated using the formula: %Specific lyses=[(Target cells^(+T cells)−Targetcells^(+BsAb+T cells))]/Target cells^(+T cells))×100%. The average IC50is 45.14 pM. FIG. 10B: T cells were activated in the killing assay asindicated by the upregulation of CD25 and CD69. FIG. 10C: cytokinesreleased by T cells in the same killing assay were quantified by ELISA.FIG. 10D: the killing potency was compared when long-term killing assaywith MV4-11 cells was performed at different E:T ratio and differentantibody concentration. FIG. 10E: anti-IL1RAP/CD3 bispecific antibodyinduced specific lysis of CD34+ enriched AML cells in the long-termkilling assay with T cells from healthy donor at E:T ratio of 1:1.Cytokines, TNF-α (FIG. 10F), IFN-r (FIG. 10G), and Granzyme B (FIG. 10H)were quantified by ELISA. FIG. 10I: long-term killing assay with AMLprimary samples using autologous T cells was conducted. In this sample,the T cell percentage is 8.97%. Two more samples were tested (FIG. 14 )FIG. 10J: anti-CD19/CD3 isotype control showed baseline killing in theautologous T cell killing assay. FIG. 10K: addition of recombinantIL1RAP in the long-term killing assay showed no significant interferenceof the killing potency for anti-IL1RAP/CD3 bispecific antibody.

FIGS. 11A-11D. MM001 induced minimal toxicity to CD34+ enriched normalbone marrow cells. FIG. 11A: IL1RAP, CD123 and CD33 expression on CD34+enriched cells from normal bone marrow. FIG. 11B: 1×10⁴ CD34+ normalbone marrow cells from three different healthy donor were mixed withautologous T cells at E:T ratio of 1:1 in the addition of MM001 atdifferent concentrations (3.15 nM, 1.05 nM, and 0 nM) for 24 hours. Thenone fourth of the cells were preceded for colony forming assay. Thecolony numbers were compared. The specific killing of CD34+ enrichednormal bone marrow cells by MM001 (FIG. 11C) and isotype control (FIG.11D) were determined by 48 hours long-term killing assay.

FIGS. 12A-12D. In vivo efficacy study of MM001. FIG. 12A; experimentdesign. 1×10⁶ MV4-11 cells were injected to each mouse via tail vein.Treatment started on day 7 with 200 ug bispecific antibody and 3×10⁶ Tcell per mouse. The subsequent treatment and imaging time were indictedby the arrow. FIG. 12B: tumor growth was monitored by bioluminescenceimaging. Massive infiltration of leukemia was seen in the NSG micetransplanted with MV4-11^(Luci) cells treated with vehicle, T cellsonly, or control BsAb plus T cells. FIG. 12C: Kaplan-Meier analysis ofsurvival for each group (n=5). Notably, mice treated with T cells hadalso a shorter survival than the control mice, probably due to thetoxicity of T cells. Mice treated with BsAb were losing weight, likelydue to the cytokine release. FIG. 12D: bioluminescent signal for eachtreatment group over time.

FIGS. 13A-13F. Expression of candidate AML immunotherapy targets on AMLcells. IL1RAP (FIG. 13A), CD123 (FIG. 13B), and CD33 (FIG. 13C)expression on different AML cells lines were detected by flow cytometry.The specific binding copies per cell (ABC or antibody binding copies) onAML cell lines were determined for IL1RAP (FIG. 13D), CD123 (FIG. 13E),and CD33 (FIG. 13F).

FIGS. 14A-14I. Killing of AML primary samples by autologous T cellsinduced by MM001. T cell percentage was 8.97% in AML 2224 (FIG. 14A),4.35% in AML 2515 (FIG. 14D), 3.05% in AML 3448 (FIG. 14G). IL1RAP+ cellpercentage was 87.1% in AML 2224 (FIG. 14B), 94.2% in AML 2515 (FIG.14E), 95.9% in AML 3448 (FIG. 14H). The killing assays were conducted byadding MM001 of indicated concentration in AML samples. Specific lysiswas detected on day 4, 6, and 8. The actual E:T ratios were 1:10 for AML2224 (FIG. 14C), 1:21.9 for AML 2515 (FIG. 14F), 1:31.8 for AML 3448(FIG. 14I).

FIGS. 15A-15D. Design and efficacy study of IL1RAP targeting CAR-T cells(CM001). 15A: schematic diagram of the CAR containing a CD8 hindgetransmembrane domain, and intracellular signaling domain of 4-1BB, andthe CD3z signaling domain. The killing potency of CM001 was conducted bylong-term killing assay with MV4-11 cells (15B), HL60 (15C), and KG-1a(15D) at different E:T ratio.

FIGS. 16A-16D. Efficacy evaluation of CM001 with CD34+ enriched AMLcells. 16A: killing of CD34+ 886 AML cells by IL1RAP-specific CAR-Tcells at indicated E:T ratio. Cytokines were determined by ELISA forIFN-γ (Panel 1), TNF-α (Panel 2), and Granzyme B (Panel 3). 16B: killingof CD34+ 2360AML cells by IL1RAP-specific CAR-T cells at indicated E:Tratio. Cytokines were determined by ELISA for IFN-γ (Panel 1),TNF-α(Panel 2), and Granzyme B (Panel 3). 16C: killing of CD34+ 2568 AMLcells by IL1RAP-specific CAR-T cells at indicated E:T ratio. Cytokineswere determined by ELISA for IFN-γ (Panel 1), TNF-α (Panel 2), andGranzyme B (Panel 3). 16D: killing of CD34+ 1003 AML cells byIL1RAP-specific CAR-T cells at indicated E:T ratio. Cytokines weredetermined by ELISA for IFN-γ (Panel 1), TNF-α(Panel 2), and Granzyme B(Panel 3).

DETAILED DESCRIPTION OF THE INVENTION

While various embodiments and aspects of the present invention are shownand described herein, it will be obvious to those skilled in the artthat such embodiments and aspects are provided by way of example only.Numerous variations, changes, and substitutions will now occur to thoseskilled in the art without departing from the invention. It should beunderstood that various alternatives to the embodiments of the inventiondescribed herein may be employed in practicing the invention.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.All documents, or portions of documents, cited in the applicationincluding, without limitation, patents, patent applications, articles,books, manuals, and treatises are hereby expressly incorporated byreference in their entirety for any purpose.

Definitions

Unless defined otherwise, technical and scientific terms used hereinhave the same meaning as commonly understood by a person of ordinaryskill in the art. See, e.g., Singleton et al., DICTIONARY OFMICROBIOLOGY AND MOLECULAR BIOLOGY 2nd ed., J. Wiley & Sons (New York,N.Y. 1994); Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL,Cold Springs Harbor Press (Cold Springs Harbor, N.Y. 1989). Any methods,devices and materials similar or equivalent to those described hereincan be used in the practice of this invention. The following definitionsare provided to facilitate understanding of certain terms usedfrequently herein and are not meant to limit the scope of the presentdisclosure.

“Nucleic acid” refers to deoxyribonucleotides or ribonucleotides andpolymers thereof in either single- or double-stranded form, andcomplements thereof. The term “polynucleotide” refers to a linearsequence of nucleotides. The term “nucleotide” typically refers to asingle unit of a polynucleotide, i.e., a monomer. Nucleotides can beribonucleotides, deoxyribonucleotides, or modified versions thereof.Examples of polynucleotides contemplated herein include single anddouble stranded DNA, single and double stranded RNA (including siRNA),and hybrid molecules having mixtures of single and double stranded DNAand RNA. Nucleic acid as used herein also refers to nucleic acids thathave the same basic chemical structure as a naturally occurring nucleicacid. Such analogues have modified sugars and/or modified ringsubstituents, but retain the same basic chemical structure as thenaturally occurring nucleic acid. A nucleic acid mimetic refers tochemical compounds that have a structure that is different the generalchemical structure of a nucleic acid, but that functions in a mannersimilar to a naturally occurring nucleic acid. Examples of suchanalogues include, without limitation, phosphorothioates,phosphoramidates, methyl phosphonates, chiral-methyl phosphonates,2-O-methyl ribonucleotides, and peptide-nucleic acids (PNAs).

The term “amino acid” refers to naturally occurring and synthetic aminoacids, as well as amino acid analogs and amino acid mimetics thatfunction in a manner similar to the naturally occurring amino acids.Naturally occurring amino acids are those encoded by the genetic code,as well as those amino acids that are later modified, e.g.,hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acidanalogs refers to compounds that have the same basic chemical structureas a naturally occurring amino acid, i.e., an α carbon that is bound toa hydrogen, a carboxyl group, an amino group, and an R group, e.g.,homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino acid. Amino acid mimetics refers tochemical compounds that have a structure that is different from thegeneral chemical structure of an amino acid, but that functions in amanner similar to a naturally occurring amino acid.

Amino acids may be referred to herein by either their commonly knownthree letter symbols or by the one-letter symbols recommended by theIUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise,may be referred to by their commonly accepted single-letter codes.

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical mimetic of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers and non-naturally occurring amino acid polymer.

An amino acid or nucleotide base “position” is denoted by a number thatsequentially identifies each amino acid (or nucleotide base) in thereference sequence based on its position relative to the N-terminus (or5′-end). Due to deletions, insertions, truncations, fusions, and thelike that may be taken into account when determining an optimalalignment, in general the amino acid residue number in a test sequencedetermined by simply counting from the N-terminus will not necessarilybe the same as the number of its corresponding position in the referencesequence. For example, in a case where a variant has a deletion relativeto an aligned reference sequence, there will be no amino acid in thevariant that corresponds to a position in the reference sequence at thesite of deletion. Where there is an insertion in an aligned referencesequence, that insertion will not correspond to a numbered amino acidposition in the reference sequence. In the case of truncations orfusions there can be stretches of amino acids in either the reference oraligned sequence that do not correspond to any amino acid in thecorresponding sequence.

The terms “numbered with reference to” or “corresponding to,” when usedin the context of the numbering of a given amino acid or polynucleotidesequence, refers to the numbering of the residues of a specifiedreference sequence when the given amino acid or polynucleotide sequenceis compared to the reference sequence. An amino acid residue in aprotein “corresponds” to a given residue when it occupies the sameessential structural position within the protein as the given residue.For example, a selected residue in a selected antibody (or Fab domain)corresponds to light chain threonine at Kabat position 40, when theselected residue occupies the same essential spatial or other structuralrelationship as a light chain threonine at Kabat position 40. In someembodiments, where a selected protein is aligned for maximum homologywith the light chain of an antibody (or Fab domain), the position in thealigned selected protein aligning with threonine 40 is said tocorrespond to threonine 40. Instead of a primary sequence alignment, athree dimensional structural alignment can also be used, e.g., where thestructure of the selected protein is aligned for maximum correspondencewith the light chain threonine at Kabat position 40, and the overallstructures compared. In this case, an amino acid that occupies the sameessential position as threonine 40 in the structural model is said tocorrespond to the threonine 40 residue.

“Conservatively modified variants” applies to both amino acid andnucleic acid sequences. With respect to particular nucleic acidsequences, “conservatively modified variants” refers to those nucleicacids that encode identical or essentially identical amino acidsequences. Because of the degeneracy of the genetic code, a number ofnucleic acid sequences will encode any given protein. For instance, thecodons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, atevery position where an alanine is specified by a codon, the codon canbe altered to any of the corresponding codons described without alteringthe encoded polypeptide. Such nucleic acid variations are “silentvariations,” which are one species of conservatively modifiedvariations. Every nucleic acid sequence herein which encodes apolypeptide also describes every possible silent variation of thenucleic acid. One of skill will recognize that each codon in a nucleicacid (except AUG, which is ordinarily the only codon for methionine, andTGG, which is ordinarily the only codon for tryptophan) can be modifiedto yield a functionally identical molecule. Accordingly, each silentvariation of a nucleic acid which encodes a polypeptide is implicit ineach described sequence.

As to amino acid sequences, one of skill will recognize that individualsubstitutions, deletions or additions to a nucleic acid, peptide,polypeptide, or protein sequence which alters, adds or deletes a singleamino acid or a small percentage of amino acids in the encoded sequenceis a “conservatively modified variant” where the alteration results inthe substitution of an amino acid with a chemically similar amino acid.Conservative substitution tables providing functionally similar aminoacids are well known in the art. Such conservatively modified variantsare in addition to and do not exclude polymorphic variants, interspecieshomologs, and alleles of the invention.

The following eight groups each contain amino acids that areconservative substitutions for one another:

1) Alanine (A), Glycine (G);

2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5)Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6)Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S),Threonine (T); and 8) Cysteine (C), Methionine (M)

(see, e.g., Creighton, Proteins (1984)).

The terms “identical” or percent “identity,” in the context of two ormore nucleic acids or polypeptide sequences, refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides that are the same(i.e., 60% identity, optionally 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%,or 99% identity over a specified region, e.g., of the entire polypeptidesequences of the invention or individual domains of the polypeptides ofthe invention), when compared and aligned for maximum correspondenceover a comparison window, or designated region as measured using one ofthe following sequence comparison algorithms or by manual alignment andvisual inspection. Such sequences are then said to be “substantiallyidentical.” This definition also refers to the complement of a testsequence. Optionally, the identity exists over a region that is at leastabout 50 nucleotides in length, or more preferably over a region that is100 to 500 or 1000 or more nucleotides in length.

“Percentage of sequence identity” is determined by comparing twooptimally aligned sequences over a comparison window, wherein theportion of the polynucleotide or polypeptide sequence in the comparisonwindow may comprise additions or deletions (i.e., gaps) as compared tothe reference sequence (which does not comprise additions or deletions)for optimal alignment of the two sequences. The percentage is calculatedby determining the number of positions at which the identical nucleicacid base or amino acid residue occurs in both sequences to yield thenumber of matched positions, dividing the number of matched positions bythe total number of positions in the window of comparison andmultiplying the result by 100 to yield the percentage of sequenceidentity.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. Default programparameters can be used, or alternative parameters can be designated. Thesequence comparison algorithm then calculates the percent sequenceidentities for the test sequences relative to the reference sequence,based on the program parameters.

A “comparison window”, as used herein, includes reference to a segmentof any one of the number of contiguous positions selected from the groupconsisting of, e.g., a full length sequence or from 20 to 600, about 50to about 200, or about 100 to about 150 amino acids or nucleotides inwhich a sequence may be compared to a reference sequence of the samenumber of contiguous positions after the two sequences are optimallyaligned. Methods of alignment of sequences for comparison are well-knownin the art. Optimal alignment of sequences for comparison can beconducted, e.g., by the local homology algorithm of Smith and Waterman(1970) Adv. Appl. Math. 2:482c, by the homology alignment algorithm ofNeedleman and Wunsch (1970) J. Mol. Biol. 48:443, by the search forsimilarity method of Pearson and Lipman (1988) Proc. Nat'l. Acad. Sci.USA 85:2444, by computerized implementations of these algorithms (GAP,BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package,Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by manualalignment and visual inspection (see, e.g., Ausubel et al., CurrentProtocols in Molecular Biology (1995 supplement)).

An example of an algorithm that is suitable for determining percentsequence identity and sequence similarity are the BLAST and BLAST 2.0algorithms, which are described in Altschul et al. (1977) Nuc. AcidsRes. 25:3389-3402, and Altschul et al. (1990) J. Mol. Biol. 215:403-410,respectively. Software for performing BLAST analyses is publiclyavailable through the National Center for Biotechnology Information.This algorithm involves first identifying high scoring sequence pairs(HSPs) by identifying short words of length W in the query sequence,which either match or satisfy some positive-valued threshold score Twhen aligned with a word of the same length in a database sequence. T isreferred to as the neighborhood word score threshold (Altschul et al.,supra). These initial neighborhood word hits act as seeds for initiatingsearches to find longer HSPs containing them. The word hits are extendedin both directions along each sequence for as far as the cumulativealignment score can be increased. Cumulative scores are calculatedusing, for nucleotide sequences, the parameters M (reward score for apair of matching residues; always >0) and N (penalty score formismatching residues; always <0). For amino acid sequences, a scoringmatrix is used to calculate the cumulative score. Extension of the wordhits in each direction are halted when: the cumulative alignment scorefalls off by the quantity X from its maximum achieved value; thecumulative score goes to zero or below, due to the accumulation of oneor more negative-scoring residue alignments; or the end of eithersequence is reached. The BLAST algorithm parameters W, T, and Xdetermine the sensitivity and speed of the alignment. The BLASTN program(for nucleotide sequences) uses as defaults a word length (W) of 11, anexpectation (E) or 10, M=5, N=−4 and a comparison of both strands. Foramino acid sequences, the BLASTP program uses as defaults a word lengthof 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (seeHenikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915)alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and a comparisonof both strands.

The BLAST algorithm also performs a statistical analysis of thesimilarity between two sequences (see, e.g., Karlin and Altschul (1993)Proc. Natl. Acad. Sci. USA 90:5873-5787). One measure of similarityprovided by the BLAST algorithm is the smallest sum probability (P(N)),which provides an indication of the probability by which a match betweentwo nucleotide or amino acid sequences would occur by chance. Forexample, a nucleic acid is considered similar to a reference sequence ifthe smallest sum probability in a comparison of the test nucleic acid tothe reference nucleic acid is less than about 0.2, more preferably lessthan about 0.01, and most preferably less than about 0.001.

An indication that two nucleic acid sequences or polypeptides aresubstantially identical is that the polypeptide encoded by the firstnucleic acid is immunologically cross reactive with the antibodiesraised against the polypeptide encoded by the second nucleic acid, asdescribed below. Thus, a polypeptide is typically substantiallyidentical to a second polypeptide, for example, where the two peptidesdiffer only by conservative substitutions. Another indication that twonucleic acid sequences are substantially identical is that the twomolecules or their complements hybridize to each other under stringentconditions, as described below. Yet another indication that two nucleicacid sequences are substantially identical is that the same primers canbe used to amplify the sequence.

Antibodies are large, complex molecules (molecular weight of ˜150,000 orabout 1320 amino acids) with intricate internal structure. A naturalantibody molecule contains two identical pairs of polypeptide chains,each pair having one light chain and one heavy chain. Each light chainand heavy chain in turn consists of two regions: a variable (“V”)region, involved in binding the target antigen, and a constant (“C”)region that interacts with other components of the immune system. Thelight and heavy chain variable regions (also referred to herein as lightchain variable (VL) domain and heavy chain variable (VH) domain,respectively) come together in 3-dimensional space to form a variableregion that binds the antigen (for example, a receptor on the surface ofa cell). Within each light or heavy chain variable region, there arethree short segments (averaging 10 amino acids in length) called thecomplementarity determining regions (“CDRs”). The six CDRs in anantibody variable domain (three from the light chain and three from theheavy chain) fold up together in 3-dimensional space to form the actualantibody binding site which docks onto the target antigen. The positionand length of the CDRs have been precisely defined by Kabat, E. et al.,Sequences of Proteins of Immunological Interest, U.S. Department ofHealth and Human Services, 1983, 1987. The part of a variable region notcontained in the CDRs is called the framework (“FR”), which forms theenvironment for the CDRs.

An “antibody variant” as provided herein refers to a polypeptide capableof binding to an antigen and including one or more structural domains ofan antibody or fragment thereof. Non-limiting examples of antibodyvariants include single-domain antibodies or nanobodies, affibodies(polypeptides smaller than monoclonal antibodies (e.g., about 6 kDA) andcapable of binding antigens with high affinity and imitating monoclonalantibodies, monospecific Fab₂, bispecific Fab₂, trispecific Fab₃,monovalent IgGs, scFv, bispecific diabodies, trispecific triabodies,scFv-Fc, minibodies, IgNAR, V-NAR, hcIgG, VhH, or peptibodies. A“nanobody” or “single domain antibody” as described herein is commonlywell known in the art and refers to an antibody fragment consisting of asingle monomeric variable antibody domain. Like a whole antibody, it isable to bind selectively to a specific antigen. A “peptibody” asprovided herein refers to a peptide moiety attached (through a covalentor non-covalent linker) to the Fc domain of an antibody. Furthernon-limiting examples of antibody variants known in the art includeantibodies produced by cartilaginous fish or camelids. A generaldescription of antibodies from camelids and the variable regions thereofand methods for their production, isolation, and use may be found inreferences WO97/49805 and WO 97/49805, which are incorporated, byreference herein in their entirety and for all purposes. Likewise,antibodies from cartilaginous fish and the variable regions thereof andmethods for their production, isolation, and use may be found inWO2005/118629, which is incorporated by reference herein in its entiretyand for all purposes.

The terms “CDR L1”, “CDR L2” and “CDR L3” as provided herein refer tothe complementarity determining regions (CDR) 1, 2, and 3 of thevariable light (L) chain of an antibody. In embodiments, the variablelight chain provided herein includes in N-terminal to C-terminaldirection a CDR L1, a CDR L2 and a CDR L3. Likewise, the terms “CDR H1”,“CDR H2” and “CDR H3” as provided herein refer to the complementaritydetermining regions (CDR) 1, 2, and 3 of the variable heavy (H) chain ofan antibody. In embodiments, the variable heavy chain provided hereinincludes in N-terminal to C-terminal direction a CDR H1, a CDR H2 and aCDR H3.

The terms “FR L1”, “FR L2”, “FR L3” and “FR L4” as provided herein areused according to their common meaning in the art and refer to theframework regions (FR) 1, 2, 3 and 4 of the variable light (L) chain ofan antibody. In embodiments, the variable light chain provided hereinincludes in N-terminal to C-terminal direction a FR L1, a FR L2, a FR L3and a FR L4. Likewise, the terms “FR H1”, “FR H2”, “FR H3” and “FR H4”as provided herein are used according to their common meaning in the artand refer to the framework regions (FR) 1, 2, 3 and 4 of the variableheavy (H) chain of an antibody. In embodiments, the variable heavy chainprovided herein includes in N-terminal to C-terminal direction a FR H1,a FR H2, a FR H3 and a FR H4.

An exemplary immunoglobulin (antibody) structural unit comprises atetramer. Each tetramer is composed of two identical pairs ofpolypeptide chains, each pair having one “light” (about 25 kD) and one“heavy” chain (about 50-70 kD). The N-terminus of each chain defines avariable region of about 100 to 110 or more amino acids primarilyresponsible for antigen recognition. The terms variable light chain(VL), variable light chain (VL) domain or light chain variable regionand variable heavy chain (VH), variable heavy chain (VH) domain or heavychain variable region refer to these light and heavy chain regions,respectively. The terms variable light chain (VL), variable light chain(VL) domain and light chain variable region as referred to herein may beused interchangeably. The terms variable heavy chain (VH), variableheavy chain (VH) domain and heavy chain variable region as referred toherein may be used interchangeably. The Fc (i.e. fragment crystallizableregion) is the “base” or “tail” of an immunoglobulin and is typicallycomposed of two heavy chains that contribute two or three constantdomains depending on the class of the antibody. By binding to specificproteins, the Fc region ensures that each antibody generates anappropriate immune response for a given antigen. The Fc region alsobinds to various cell receptors, such as Fc receptors, and other immunemolecules, such as complement proteins.

The term “antibody” is used according to its commonly known meaning inthe art. Antibodies exist, e.g., as intact immunoglobulins or as anumber of well-characterized fragments produced by digestion withvarious peptidases. Thus, for example, pepsin digests an antibody belowthe disulfide linkages in the hinge region to produce F(ab)′₂, a dimerof Fab which itself is a light chain joined to V_(H)-C_(H1) by adisulfide bond. The F(ab)′₂ may be reduced under mild conditions tobreak the disulfide linkage in the hinge region, thereby converting theF(ab)′₂ dimer into an Fab′ monomer. The Fab′ monomer is essentially Fabwith part of the hinge region (see Fundamental Immunology (Paul ed., 3ded. 1993). While various antibody fragments are defined in terms of thedigestion of an intact antibody, one of skill will appreciate that suchfragments may be synthesized de novo either chemically or by usingrecombinant DNA methodology. Thus, the term antibody, as used herein,also includes antibody fragments either produced by the modification ofwhole antibodies, or those synthesized de novo using recombinant DNAmethodologies (e.g., single chain Fv) or those identified using phagedisplay libraries (see, e.g., McCafferty et al., Nature 348:552-554(1990)).

For preparation of monoclonal or polyclonal antibodies, any techniqueknown in the art can be used (see, e.g., Kohler & Milstein, Nature256:495-497 (1975); Kozbor et al., Immunology Today 4:72 (1983); Cole etal., pp. 77-96 in Monoclonal Antibodies and Cancer Therapy (1985)).“Monoclonal” antibodies (mAb) refer to antibodies derived from a singleclone. Techniques for the production of single chain antibodies (U.S.Pat. No. 4,946,778) can be adapted to produce antibodies to polypeptidesof this invention. Also, transgenic mice, or other organisms such asother mammals, may be used to express humanized antibodies.Alternatively, phage display technology can be used to identifyantibodies and heteromeric Fab fragments that specifically bind toselected antigens (see, e.g., McCafferty et al., Nature 348:552-554(1990); Marks et al., Biotechnology 10:779-783 (1992)).

The epitope of a mAb is the region of its antigen to which the mAbbinds. Two antibodies bind to the same or overlapping epitope if eachcompetitively inhibits (blocks) binding of the other to the antigen.That is, a 1×, 5×, 10×, 20× or 100× excess of one antibody inhibitsbinding of the other by at least 30% but preferably 50%, 75%, 90% oreven 99% as measured in a competitive binding assay (see, e.g., Junghanset al., Cancer Res. 50:1495, 1990). Alternatively, two antibodies havethe same epitope if essentially all amino acid mutations in the antigenthat reduce or eliminate binding of one antibody reduce or eliminatebinding of the other. Two antibodies have overlapping epitopes if someamino acid mutations that reduce or eliminate binding of one antibodyreduce or eliminate binding of the other.

A single-chain variable fragment (scFv) is typically a fusion protein ofthe variable regions of the heavy (VH) and light chains (VL) ofimmunoglobulins, connected with a short linker peptide of 10 to about 25amino acids. The linker may usually be rich in glycine for flexibility,as well as serine or threonine for solubility. The linker can eitherconnect the N-terminus of the VH with the C-terminus of the VL, or viceversa. In embodiments, the linker includes more than one serine. Inembodiments, the linker includes more than one glycine. In embodiments,the linker has the structure of -(Gly-Gly-Gly-Gly-Ser)₃- (SEQ ID NO:19).

For preparation of suitable antibodies of the invention and for useaccording to the invention, e.g., recombinant, monoclonal, or polyclonalantibodies, many techniques known in the art can be used (see, e.g.,Kohler & Milstein, Nature 256:495-497 (1975); Kozbor et al., ImmunologyToday 4: 72 (1983); Cole et al., pp. 77-96 in Monoclonal Antibodies andCancer Therapy, Alan R. Liss, Inc. (1985); Coligan, Current Protocols inImmunology (1991); Harlow & Lane, Antibodies, A Laboratory Manual(1988); and Goding, Monoclonal Antibodies: Principles and Practice (2ded. 1986)). The genes encoding the heavy and light chains of an antibodyof interest can be cloned from a cell, e.g., the genes encoding amonoclonal antibody can be cloned from a hybridoma and used to produce arecombinant monoclonal antibody. Gene libraries encoding heavy and lightchains of monoclonal antibodies can also be made from hybridoma orplasma cells. Random combinations of the heavy and light chain geneproducts generate a large pool of antibodies with different antigenicspecificity (see, e.g., Kuby, Immunology (3rd ed. 1997)). Techniques forthe production of single chain antibodies or recombinant antibodies(U.S. Pat. Nos. 4,946,778, 4,816,567) can be adapted to produceantibodies to polypeptides of this invention. Also, transgenic mice, orother organisms such as other mammals, may be used to express humanizedor human antibodies (see, e.g., U.S. Pat. Nos. 5,545,807; 5,545,806;5,569,825; 5,625,126; 5,633,425; 5,661,016, Marks et al., Bio/Technology10:779-783 (1992); Lonberg et al., Nature 368:856-859 (1994); Morrison,Nature 368:812-13 (1994); Fishwild et al., Nature Biotechnology14:845-51 (1996); Neuberger, Nature Biotechnology 14:826 (1996); andLonberg & Huszar, Intern. Rev. Immunol. 13:65-93 (1995)). Alternatively,phage display technology can be used to identify antibodies andheteromeric Fab fragments that specifically bind to selected antigens(see, e.g., McCafferty et al., Nature 348:552-554 (1990); Marks et al.,Biotechnology 10:779-783 (1992)). Antibodies can also be madebispecific, i.e., able to recognize two different antigens (see, e.g.,WO 93/08829, Traunecker et al., EMBO J. 10:3655-3659 (1991); and Sureshet al., Methods in Enzymology 121:210 (1986)). Antibodies can also beheteroconjugates, e.g., two covalently joined antibodies, orimmunotoxins (see, e.g., U.S. Pat. No. 4,676,980, WO 91/00360; WO92/200373; and EP 03089).

Methods for humanizing or primatizing non-human antibodies are wellknown in the art (e.g., U.S. Pat. Nos. 4,816,567; 5,530,101; 5,859,205;5,585,089; 5,693,761; 5,693,762; 5,777,085; 6,180,370; 6,210,671; and6,329,511; WO 87/02671; EP Patent Application 0173494; Jones et al.(1986) Nature 321:522; and Verhoyen et al. (1988) Science 239:1534).Humanized antibodies are further described in, e.g., Winter and Milstein(1991) Nature 349:293. Generally, a humanized antibody has one or moreamino acid residues introduced into it from a source which is non-human.These non-human amino acid residues are often referred to as importresidues, which are typically taken from an import variable domain.Humanization can be essentially performed following the method of Winterand co-workers (see, e.g., Morrison et al., PNAS USA, 81:6851-6855(1984), Jones et al., Nature 321:522-525 (1986); Riechmann et al.,Nature 332:323-327 (1988); Morrison and Oi, Adv. Immunol., 44:65-92(1988), Verhoeyen et al., Science 239:1534-1536 (1988) and Presta, Curr.Op. Struct. Biol. 2:593-596 (1992), Padlan, Molec. Immun., 28:489-498(1991); Padlan, Molec. Immun., 31(3):169-217 (1994)), by substitutingrodent CDRs or CDR sequences for the corresponding sequences of a humanantibody. Accordingly, such humanized antibodies are chimeric antibodies(U.S. Pat. No. 4,816,567), wherein substantially less than an intacthuman variable domain has been substituted by the corresponding sequencefrom a non-human species. In practice, humanized antibodies aretypically human antibodies in which some CDR residues and possibly someFR residues are substituted by residues from analogous sites in rodentantibodies. For example, polynucleotides comprising a first sequencecoding for humanized immunoglobulin framework regions and a secondsequence set coding for the desired immunoglobulin complementaritydetermining regions can be produced synthetically or by combiningappropriate cDNA and genomic DNA segments. Human constant region DNAsequences can be isolated in accordance with well known procedures froma variety of human cells.

A “chimeric antibody” is an antibody molecule in which (a) the constantregion, or a portion thereof, is altered, replaced or exchanged so thatthe antigen binding site (variable region) is linked to a constantregion of a different or altered class, effector function and/orspecies, or an entirely different molecule which confers new propertiesto the chimeric antibody, e.g., an enzyme, toxin, hormone, growthfactor, drug, etc.; or (b) the variable region, or a portion thereof, isaltered, replaced or exchanged with a variable region having a differentor altered antigen specificity. The preferred antibodies of, and for useaccording to the invention include humanized and/or chimeric monoclonalantibodies.

Techniques for conjugating therapeutic agents to antibodies are wellknown (see, e.g., Arnon et al., “Monoclonal Antibodies ForImmunotargeting Of Drugs In Cancer Therapy”, in Monoclonal AntibodiesAnd Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss,Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery” inControlled Drug Delivery (2^(nd) Ed.), Robinson et al. (eds.), pp.623-53 (Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers OfCytotoxic Agents In Cancer Therapy: A Review” in Monoclonal Antibodies'84: Biological And Clinical Applications, Pinchera et al. (eds.), pp.475-506 (1985); and Thorpe et al., “The Preparation And CytotoxicProperties Of Antibody-Toxin Conjugates”, Immunol. Rev., 62:119-58(1982)). As used herein, the term “antibody-drug conjugate” or “ADC”refers to a therapeutic agent conjugated or otherwise covalently boundto to an antibody.

A “therapeutic agent” as referred to herein, is a composition useful intreating or preventing a disease such as cancer (e.g., leukemia). Inembodiments, the therpaeutic agent is an anti-cancer agent. “Anti-canceragent” is used in accordance with its plain ordinary meaning and refersto a composition (e.g. compound, drug, antagonist, inhibitor, modulator)having antineoplastic properties or the ability to inhibit the growth orproliferation of cells. In embodiments, an anti-cancer agent is achemotherapeutic. In embodiments, an anti-cancer agent is an agentidentified herein having utility in methods of treating cancer. Inembodiments, an anti-cancer agent is an agent approved by the FDA orsimilar regulatory agency of a country other than the USA, for treatingcancer.

The phrase “specifically (or selectively) binds” to an antibody or“specifically (or selectively) immunoreactive with,” when referring to aprotein or peptide, refers to a binding reaction that is determinativeof the presence of the protein, often in a heterogeneous population ofproteins and other biologics. Thus, under designated immunoassayconditions, the specified antibodies bind to a particular protein atleast two times the background and more typically more than 10 to 100times background. Specific binding to an antibody under such conditionsrequires an antibody that is selected for its specificity for aparticular protein. For example, polyclonal antibodies can be selectedto obtain only a subset of antibodies that are specificallyimmunoreactive with the selected antigen and not with other proteins.This selection may be achieved by subtracting out antibodies thatcross-react with other molecules. A variety of immunoassay formats maybe used to select antibodies specifically immunoreactive with aparticular protein. For example, solid-phase ELISA immunoassays areroutinely used to select antibodies specifically immunoreactive with aprotein (see, e.g., Harlow & Lane, Using Antibodies, A Laboratory Manual(1998) for a description of immunoassay formats and conditions that canbe used to determine specific immunoreactivity).

A “ligand” refers to an agent, e.g., a polypeptide or other molecule,capable of binding to a receptor or antibody, antibody variant, antibodyregion or fragment thereof.

The term “IL1RAP” as used herein refers to any recombinant ornaturally-occurring forms of interleukin-1 receptor accessory protein(IL1RAP) or variants or homologs thereof that maintain IL1RAP activity(e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100%activity compared to IL1RAP). In some aspects, the variants or homologshave at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequenceidentity across the whole sequence or a portion of the sequence (e.g. a10, 20, 50, 100, 150 or 200 continuous amino acid portion) compared to anaturally occurring IL1RAP polypeptide. In embodiments, IL1RAP issubstantially identical to the protein identified by the UniProtreference number Q9NPH3 or a variant or homolog having substantialidentity thereto.

A “label” or a “detectable moiety” is a composition detectable byspectroscopic, photochemical, biochemical, immunochemical, chemical, orother physical means. For example, useful labels include 32P,fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonlyused in an ELISA), biotin, digoxigenin, or haptens and proteins or otherentities which can be made detectable, e.g., by incorporating aradiolabel into a peptide or antibody specifically reactive with atarget peptide. Any appropriate method known in the art for conjugatingan antibody to the label may be employed, e.g., using methods describedin Hermanson, Bioconjugate Techniques 1996, Academic Press, Inc., SanDiego.

“Contacting” is used in accordance with its plain ordinary meaning andrefers to the process of allowing at least two distinct species (e.g.antibodies and antigens) to become sufficiently proximal to react,interact, or physically touch. It should be appreciated; however, thatthe resulting reaction product can be produced directly from a reactionbetween the added reagents or from an intermediate from one or more ofthe added reagents which can be produced in the reaction mixture.

The term “contacting” may include allowing two species to react,interact, or physically touch, wherein the two species may be, forexample, a pharmaceutical composition as provided herein and a cell. Inembodiments contacting includes, for example, allowing a pharmaceuticalcomposition as described herein to interact with a cell.

A “cell” as used herein, refers to a cell carrying out metabolic orother function sufficient to preserve or replicate its genomic DNA. Acell can be identified by well-known methods in the art including, forexample, presence of an intact membrane, staining by a particular dye,ability to produce progeny or, in the case of a gamete, ability tocombine with a second gamete to produce a viable offspring. Cells mayinclude prokaryotic and eukaryotic cells. Prokaryotic cells include butare not limited to bacteria. Eukaryotic cells include, but are notlimited to, yeast cells and cells derived from plants and animals, forexample mammalian, insect (e.g., spodoptera) and human cells.

A “stem cell” as provided herein refers to a cell characterized by theability of self-renewal through mitotic cell division and the potentialto differentiate into a tissue or an organ. Among mammalian stem cells,embryonic stem cells (ES cells) and somatic stem cells (e.g., HSC) canbe distinguished. Embryonic stem cells reside in the blastocyst and giverise to embryonic tissues, whereas somatic stem cells reside in adulttissues for the purpose of tissue regeneration and repair. Inembodiments, the stem cell is a leukemia stem cell (LSC). A “leukemiastem cell or “LSC” as provided herein refers to a cell capable ofinitiating the disease (leukemia) when transplanted into immunodeficientanimals and can self-renew by giving rise to leukemia in serialtransplantations and also partially differentiate into non-LSC bulkblasts that resemble the original disease but are unable to self-renew.An LSC may carry a gene mutation and be able to self-renew throughmitotic cell division and differentiate into the hematopoietic lineagecarrying said gene mutant or an LSC may remain as immature progenitorcells, also known as blast cells. In embodiments, the LSC expressesCD34.

The term “CD34” as referred to herein includes any of the recombinant ornaturally-occurring forms of the cluster of differentiation 34 protein,or variants or homologs thereof that maintain CD34 activity (e.g. withinat least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activitycompared to CD34). In some aspects, the variants or homologs have atleast 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identityacross the whole sequence or a portion of the sequence (e.g. a 50, 100,150 or 200 continuous amino acid portion) compared to a naturallyoccurring CD34 protein. In embodiments, the CD34 protein issubstantially identical to the protein identified by the UniProtreference number P28906 or a variant or homolog having substantialidentity thereto.

The term “recombinant” when used with reference, e.g., to a cell,nucleic acid, protein, or vector, indicates that the cell, nucleic acid,protein or vector, has been modified by the introduction of aheterologous nucleic acid or protein or the alteration of a nativenucleic acid or protein, or that the cell is derived from a cell somodified. Thus, for example, recombinant cells express genes that arenot found within the native (non-recombinant) form of the cell orexpress native genes that are otherwise abnormally expressed, underexpressed or not expressed at all. Transgenic cells and plants are thosethat express a heterologous gene or coding sequence, typically as aresult of recombinant methods.

The term “heterologous” when used with reference to portions of anucleic acid indicates that the nucleic acid comprises two or moresubsequences that are not found in the same relationship to each otherin nature. For instance, the nucleic acid is typically recombinantlyproduced, having two or more sequences from unrelated genes arranged tomake a new functional nucleic acid, e.g., a promoter from one source anda coding region from another source. Similarly, a heterologous proteinindicates that the protein comprises two or more subsequences that arenot found in the same relationship to each other in nature (e.g., afusion protein).

The term “exogenous” refers to a molecule or substance (e.g., acompound, nucleic acid or protein) that originates from outside a givencell or organism. For example, an “exogenous promoter” as referred toherein is a promoter that does not originate from the cell or organismit is expressed by. Conversely, the term “endogenous” or “endogenouspromoter” refers to a molecule or substance that is native to, ororiginates within, a given cell or organism.

As defined herein, the term “inhibition”, “inhibit”, “inhibiting” andthe like in reference to cell proliferation (e.g., cancer cellproliferation) means negatively affecting (e.g., decreasingproliferation) or killing the cell. In some embodiments, inhibitionrefers to reduction of a disease or symptoms of disease (e.g., cancer,cancer cell proliferation). Thus, inhibition includes, at least in part,partially or totally blocking stimulation, decreasing, preventing, ordelaying activation, or inactivating, desensitizing, or down-regulatingsignal transduction or enzymatic activity or the amount of a protein.Similarly an “inhibitor” is a compound or protein that inhibits areceptor or another protein, e.g., by binding, partially or totallyblocking, decreasing, preventing, delaying, inactivating, desensitizing,or down-regulating activity (e.g., a receptor activity or a proteinactivity).

“Biological sample” or “sample” refer to materials obtained from orderived from a subject or patient. A biological sample includes sectionsof tissues such as biopsy and autopsy samples, and frozen sections takenfor histological purposes. Such samples include bodily fluids such asblood and blood fractions or products (e.g., serum, plasma, platelets,red blood cells, and the like), sputum, tissue, cultured cells (e.g.,primary cultures, explants, and transformed cells) stool, urine,synovial fluid, joint tissue, synovial tissue, synoviocytes,fibroblast-like synoviocytes, macrophage-like synoviocytes, immunecells, hematopoietic cells, fibroblasts, macrophages, T cells, etc. Abiological sample is typically obtained from a eukaryotic organism, suchas a mammal such as a primate e.g., chimpanzee or human; cow; dog; cat;a rodent, e.g., guinea pig, rat, mouse; rabbit; or a bird; reptile; orfish.

A “control” or “standard control” refers to a sample, measurement, orvalue that serves as a reference, usually a known reference, forcomparison to a test sample, measurement, or value. For example, a testsample can be taken from a patient suspected of having a given disease(e.g. cancer) and compared to a known normal (non-diseased) individual(e.g. a standard control subject). A standard control can also representan average measurement or value gathered from a population of similarindividuals (e.g. standard control subjects) that do not have a givendisease (i.e. standard control population), e.g., healthy individualswith a similar medical background, same age, weight, etc. A standardcontrol value can also be obtained from the same individual, e.g. froman earlier-obtained sample from the patient prior to disease onset. Forexample, a control can be devised to compare therapeutic benefit basedon pharmacological data (e.g., half-life) or therapeutic measures (e.g.,comparison of side effects). Controls are also valuable for determiningthe significance of data. For example, if values for a given parameterare widely variant in controls, variation in test samples will not beconsidered as significant. One of skill will recognize that standardcontrols can be designed for assessment of any number of parameters(e.g. RNA levels, protein levels, specific cell types, specific bodilyfluids, specific tissues, synoviocytes, synovial fluid, synovial tissue,fibroblast-like synoviocytes, macrophagelike synoviocytes, etc).

One of skill in the art will understand which standard controls are mostappropriate in a given situation and be able to analyze data based oncomparisons to standard control values. Standard controls are alsovaluable for determining the significance (e.g. statisticalsignificance) of data. For example, if values for a given parameter arewidely variant in standard controls, variation in test samples will notbe considered as significant.

“Patient” or “subject in need thereof” refers to a living organismsuffering from or prone to a disease or condition that can be treated byadministration of a composition or pharmaceutical composition asprovided herein. Non-limiting examples include humans, other mammals,bovines, rats, mice, dogs, monkeys, goat, sheep, cows, deer, and othernon-mammalian animals. In some embodiments, a patient is human.

The terms “disease” or “condition” refer to a state of being or healthstatus of a patient or subject capable of being treated with thecompounds or methods provided herein. The disease may be a cancer. Insome further instances, “cancer” refers to human cancers and carcinomas,sarcomas, adenocarcinomas, lymphomas, leukemias, including solid andlymphoid cancers, kidney, breast, lung, bladder, colon, ovarian,prostate, pancreas, stomach, brain, head and neck, skin, uterine,testicular, glioma, esophagus, and liver cancer, includinghepatocarcinoma, lymphoma, including B-acute lymphoblastic lymphoma,non-Hodgkin's lymphomas (e.g., Burkitt's, Small Cell, and Large Celllymphomas), Hodgkin's lymphoma, leukemia (including acute myeloidleukemia (AML), ALL, and CML), or multiple myeloma.

As used herein, the term “cancer” refers to all types of cancer,neoplasm or malignant tumors found in mammals (e.g., humans), includingleukemia, carcinomas and sarcomas. Exemplary cancers that may be treatedwith a compound or method provided herein include breast cancer, coloncancer, kidney cancer, leukemia, lung cancer, melanoma, ovarian cancer,prostate cancer, pancreatic cancer, brain cancer, liver cancer, gastriccancer or a sarcoma.

The term “leukemia” refers broadly to progressive, malignant diseases ofthe blood-forming organs and is generally characterized by a distortedproliferation and development of leukocytes and their precursors in theblood and bone marrow. Leukemia is generally clinically classified onthe basis of (1) the duration and character of the disease-acute orchronic; (2) the type of cell involved; myeloid (myelogenous), lymphoid(lymphogenous), or monocytic; and (3) the increase or non-increase inthe number abnormal cells in the blood-leukemic or aleukemic(subleukemic). Exemplary leukemias that may be treated with a compoundor method provided herein include, for example, acute myeloid leukemia,acute nonlymphocytic leukemia, chronic lymphocytic leukemia, acutegranulocytic leukemia, chronic granulocytic leukemia, acutepromyelocytic leukemia, adult T-cell leukemia, aleukemic leukemia, aleukocythemic leukemia, basophylic leukemia, blast cell leukemia, bovineleukemia, chronic myelocytic leukemia, leukemia cutis, embryonalleukemia, eosinophilic leukemia, Gross' leukemia, hairy-cell leukemia,hemoblastic leukemia, hemocytoblastic leukemia, histiocytic leukemia,stem cell leukemia, acute monocytic leukemia, leukopenic leukemia,lymphatic leukemia, lymphoblastic leukemia, lymphocytic leukemia,lymphogenous leukemia, lymphoid leukemia, lymphosarcoma cell leukemia,mast cell leukemia, megakaryocytic leukemia, micromyeloblastic leukemia,monocytic leukemia, myeloblastic leukemia, myelocytic leukemia, myeloidgranulocytic leukemia, myelomonocytic leukemia, Naegeli leukemia, plasmacell leukemia, multiple myeloma, plasmacytic leukemia, promyelocyticleukemia, Rieder cell leukemia, Schilling's leukemia, stem cellleukemia, subleukemic leukemia, or undifferentiated cell leukemia.

The term “sarcoma” generally refers to a tumor which is made up of asubstance like the embryonic connective tissue and is generally composedof closely packed cells embedded in a fibrillar or homogeneoussubstance. Sarcomas that may be treated with a compound or methodprovided herein include a chondrosarcoma, fibrosarcoma, lymphosarcoma,melanosarcoma, myxosarcoma, osteosarcoma, Abemethy's sarcoma, adiposesarcoma, liposarcoma, alveolar soft part sarcoma, ameloblastic sarcoma,botryoid sarcoma, chloroma sarcoma, chorio carcinoma, embryonal sarcoma,Wilms' tumor sarcoma, endometrial sarcoma, stromal sarcoma, Ewing'ssarcoma, fascial sarcoma, fibroblastic sarcoma, giant cell sarcoma,granulocytic sarcoma, Hodgkin's sarcoma, idiopathic multiple pigmentedhemorrhagic sarcoma, immunoblastic sarcoma of B cells, lymphoma,immunoblastic sarcoma of T-cells, Jensen's sarcoma, Kaposi's sarcoma,Kupffer cell sarcoma, angiosarcoma, leukosarcoma, malignant mesenchymomasarcoma, parosteal sarcoma, reticulocytic sarcoma, Rous sarcoma,serocystic sarcoma, synovial sarcoma, or telangiectaltic sarcoma.

The term “melanoma” is taken to mean a tumor arising from themelanocytic system of the skin and other organs. Melanomas that may betreated with a compound or method provided herein include, for example,acral-lentiginous melanoma, amelanotic melanoma, benign juvenilemelanoma, Cloudman's melanoma, S91 melanoma, Harding-Passey melanoma,juvenile melanoma, lentigo maligna melanoma, malignant melanoma, nodularmelanoma, subungal melanoma, or superficial spreading melanoma.

The term “carcinoma” refers to a malignant new growth made up ofepithelial cells tending to infiltrate the surrounding tissues and giverise to metastases. Exemplary carcinomas that may be treated with acompound or method provided herein include, for example, medullarythyroid carcinoma, familial medullary thyroid carcinoma, acinarcarcinoma, acinous carcinoma, adenocystic carcinoma, adenoid cysticcarcinoma, carcinoma adenomatosum, carcinoma of adrenal cortex, alveolarcarcinoma, alveolar cell carcinoma, basal cell carcinoma, carcinomabasocellulare, basaloid carcinoma, basosquamous cell carcinoma,bronchioalveolar carcinoma, bronchiolar carcinoma, bronchogeniccarcinoma, cerebriform carcinoma, cholangiocellular carcinoma, chorioniccarcinoma, colloid carcinoma, comedo carcinoma, corpus carcinoma,cribriform carcinoma, carcinoma en cuirasse, carcinoma cutaneum,cylindrical carcinoma, cylindrical cell carcinoma, duct carcinoma,carcinoma durum, embryonal carcinoma, encephaloid carcinoma, epiermoidcarcinoma, carcinoma epitheliale adenoides, exophytic carcinoma,carcinoma ex ulcere, carcinoma fibrosum, gelatiniforni carcinoma,gelatinous carcinoma, giant cell carcinoma, carcinoma gigantocellulare,glandular carcinoma, granulosa cell carcinoma, hair-matrix carcinoma,hematoid carcinoma, hepatocellular carcinoma, Hurthle cell carcinoma,hyaline carcinoma, hypernephroid carcinoma, infantile embryonalcarcinoma, carcinoma in situ, intraepidermal carcinoma, intraepithelialcarcinoma, Krompecher's carcinoma, Kulchitzky-cell carcinoma, large-cellcarcinoma, lenticular carcinoma, carcinoma lenticulare, lipomatouscarcinoma, lymphoepithelial carcinoma, carcinoma medullare, medullarycarcinoma, melanotic carcinoma, carcinoma molle, mucinous carcinoma,carcinoma muciparum, carcinoma mucocellulare, mucoepidermoid carcinoma,carcinoma mucosum, mucous carcinoma, carcinoma myxomatodes,nasopharyngeal carcinoma, oat cell carcinoma, carcinoma ossificans,osteoid carcinoma, papillary carcinoma, periportal carcinoma,preinvasive carcinoma, prickle cell carcinoma, pultaceous carcinoma,renal cell carcinoma of kidney, reserve cell carcinoma, carcinomasarcomatodes, schneiderian carcinoma, scirrhous carcinoma, carcinomascroti, signet-ring cell carcinoma, carcinoma simplex, small-cellcarcinoma, solanoid carcinoma, spheroidal cell carcinoma, spindle cellcarcinoma, carcinoma spongiosum, squamous carcinoma, squamous cellcarcinoma, string carcinoma, carcinoma telangiectaticum, carcinomatelangiectodes, transitional cell carcinoma, carcinoma tuberosum,tuberous carcinoma, verrucous carcinoma, or carcinoma villosum.

As used herein, the terms “metastasis,” “metastatic,” and “metastaticcancer” can be used interchangeably and refer to the spread of aproliferative disease or disorder, e.g., cancer, from one organ oranother non-adjacent organ or body part. Cancer occurs at an originatingsite, e.g., breast, which site is referred to as a primary tumor, e.g.,primary breast cancer. Some cancer cells in the primary tumor ororiginating site acquire the ability to penetrate and infiltratesurrounding normal tissue in the local area and/or the ability topenetrate the walls of the lymphatic system or vascular systemcirculating through the system to other sites and tissues in the body. Asecond clinically detectable tumor formed from cancer cells of a primarytumor is referred to as a metastatic or secondary tumor. When cancercells metastasize, the metastatic tumor and its cells are presumed to besimilar to those of the original tumor. Thus, if lung cancermetastasizes to the breast, the secondary tumor at the site of thebreast consists of abnormal lung cells and not abnormal breast cells.The secondary tumor in the breast is referred to a metastatic lungcancer. Thus, the phrase metastatic cancer refers to a disease in whicha subject has or had a primary tumor and has one or more secondarytumors. The phrases non-metastatic cancer or subjects with cancer thatis not metastatic refers to diseases in which subjects have a primarytumor but not one or more secondary tumors. For example, metastatic lungcancer refers to a disease in a subject with or with a history of aprimary lung tumor and with one or more secondary tumors at a secondlocation or multiple locations, e.g., in the breast.

The term “associated” or “associated with” in the context of a substanceor substance activity or function associated with a disease (e.g.,cancer (e.g. leukemia, acute myeloid leukemia)) means that the disease(e.g., cancer (e.g. leukemia, acute myeloid leukemia)) is caused by (inwhole or in part), or a symptom of the disease is caused by (in whole orin part) the substance or substance activity or function. Alternatively,the substance (e.g., IL1RAP) may be an indicator of the disease (e.g.,cancer (e.g. leukemia, acute myeloid leukemia)). Thus, an associatedsubstance may serve as a means of targeting disease tissue (e.g., cancercells (e.g., leukemia stem cells, acute myeloid leukemia cells)).

As used herein, “treating” or “treatment of” a condition, disease ordisorder or symptoms associated with a condition, disease (e.g., cancer,e.g., AML) or disorder refers to an approach for obtaining beneficial ordesired results, including clinical results. Beneficial or desiredclinical results can include, but are not limited to, alleviation oramelioration of one or more symptoms or conditions, diminishment ofextent of condition, disorder or disease, stabilization of the state ofcondition, disorder or disease, prevention of development of condition,disorder or disease, prevention of spread of condition, disorder ordisease, delay or slowing of condition, disorder or disease progression,delay or slowing of condition, disorder or disease onset, ameliorationor palliation of the condition, disorder or disease state, andremission, whether partial or total. “Treating” can also mean prolongingsurvival of a subject beyond that expected in the absence of treatment.“Treating” can also mean inhibiting the progression of the condition,disorder or disease, slowing the progression of the condition, disorderor disease temporarily, although in some instances, it involves haltingthe progression of the condition, disorder or disease permanently. Asused herein the terms treatment, treat, or treating refers to a methodof reducing the effects of one or more symptoms of a disease orcondition characterized by expression of the protease or symptom of thedisease or condition characterized by expression of the protease. Thusin the disclosed method, treatment can refer to a 10%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 90%, or 100% reduction in the severity of anestablished disease, condition, or symptom of the disease or condition.For example, a method for treating a disease is considered to be atreatment if there is a 10% reduction in one or more symptoms of thedisease in a subject as compared to a control. Thus the reduction can bea 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any percentreduction in between 10% and 100% as compared to native or controllevels. It is understood that treatment does not necessarily refer to acure or complete ablation of the disease, condition, or symptoms of thedisease or condition. Further, as used herein, references to decreasing,reducing, or inhibiting include a change of 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90% or greater as compared to a control level and suchterms can include but do not necessarily include complete elimination.

The terms “dose” and “dosage” are used interchangeably herein. A doserefers to the amount of active ingredient given to an individual at eachadministration. The dose will vary depending on a number of factors,including the range of normal doses for a given therapy, frequency ofadministration; size and tolerance of the individual; severity of thecondition; risk of side effects; and the route of administration. One ofskill will recognize that the dose can be modified depending on theabove factors or based on therapeutic progress. The term “dosage form”refers to the particular format of the pharmaceutical or pharmaceuticalcomposition, and depends on the route of administration. For example, adosage form can be in a liquid form for nebulization, e.g., forinhalants, in a tablet or liquid, e.g., for oral delivery, or a salinesolution, e.g., for injection.

By “therapeutically effective dose or amount” as used herein is meant adose that produces effects for which it is administered (e.g. treatingor preventing a disease). The exact dose and formulation will depend onthe purpose of the treatment, and will be ascertainable by one skilledin the art using known techniques (see, e.g., Lieberman, PharmaceuticalDosage Forms (vols. 1-3, 1992); Lloyd, The Art, Science and Technologyof Pharmaceutical Compounding (1999); Remington: The Science andPractice of Pharmacy, 20th Edition, Gennaro, Editor (2003), and Pickar,Dosage Calculations (1999)). For example, for the given parameter, atherapeutically effective amount will show an increase or decrease of atleast 5%, 10%, 15%, 20%, 25%, 40%, 50%, 60%, 75%, 80%, 90%, or at least100%. Therapeutic efficacy can also be expressed as “-fold” increase ordecrease. For example, a therapeutically effective amount can have atleast a 1.2-fold, 1.5-fold, 2-fold, 5-fold, or more effect over astandard control. A therapeutically effective dose or amount mayameliorate one or more symptoms of a disease. A therapeuticallyeffective dose or amount may prevent or delay the onset of a disease orone or more symptoms of a disease when the effect for which it is beingadministered is to treat a person who is at risk of developing thedisease.

In embodiments, the method further includes administering to the subjectan additional therapeutic agent. As described above, a therapeutic agentis a composition useful in treating or preventing a disease such ascancer. In embodiments, the additional therapeutic agent is ananti-cancer agent.

The terms “anti-cancer agent” and “anticancer agent” are used inaccordance with their plain ordinary meaning and refer to a composition(e.g. compound, drug, antagonist, inhibitor, modulator) havingantineoplastic properties or the ability to inhibit the growth orproliferation of cells. In some embodiments, an anti-cancer agent is achemotherapeutic. In some embodiments, an anti-cancer agent is an agentidentified herein having utility in methods of treating cancer. In someembodiments, an anti-cancer agent is an agent approved by the FDA orsimilar regulatory agency of a country other than the USA, for treatingcancer. Examples of anti-cancer agents include, but are not limited to,MEK (e.g. MEK1, MEK2, or MEK1 and MEK2) inhibitors (e.g. XL518, CI-1040,PD035901, selumetinib/AZD6244, GSK1120212/trametinib, GDC-0973,ARRY-162, ARRY-300, AZD8330, PD0325901, U0126, PD98059, TAK-733,PD318088, AS703026, BAY 869766), alkylating agents (e.g.,cyclophosphamide, ifosfamide, chlorambucil, busulfan, melphalan,mechlorethamine, uramustine, thiotepa, nitrosoureas, nitrogen mustards(e.g., mechloroethamine, cyclophosphamide, chlorambucil, meiphalan),ethylenimine and methylmelamines (e.g., hexamethlymelamine, thiotepa),alkyl sulfonates (e.g., busulfan), nitrosoureas (e.g., carmustine,lomusitne, semustine, streptozocin), triazenes (decarbazine)),anti-metabolites (e.g., 5-azathioprine, leucovorin, capecitabine,fludarabine, gemcitabine, pemetrexed, raltitrexed, folic acid analog(e.g., methotrexate), or pyrimidine analogs (e.g., fluorouracil,floxouridine, Cytarabine), purine analogs (e.g., mercaptopurine,thioguanine, pentostatin), etc.), plant alkaloids (e.g., vincristine,vinblastine, vinorelbine, vindesine, podophyllotoxin, paclitaxel,docetaxel, etc.), topoisomerase inhibitors (e.g., irinotecan, topotecan,amsacrine, etoposide (VP16), etoposide phosphate, teniposide, etc.),antitumor antibiotics (e.g., doxorubicin, adriamycin, daunorubicin,epirubicin, actinomycin, bleomycin, mitomycin, mitoxantrone, plicamycin,etc.), platinum-based compounds (e.g. cisplatin, oxaloplatin,carboplatin), anthracenedione (e.g., mitoxantrone), substituted urea(e.g., hydroxyurea), methyl hydrazine derivative (e.g., procarbazine),adrenocortical suppressant (e.g., mitotane, aminoglutethimide),epipodophyllotoxins (e.g., etoposide), antibiotics (e.g., daunorubicin,doxorubicin, bleomycin), enzymes (e.g., L-asparaginase), inhibitors ofmitogen-activated protein kinase signaling (e.g. U0126, PD98059,PD184352, PD0325901, ARRY-142886, SB239063, SP600125, BAY 43-9006,wortmannin, or LY294002, Syk inhibitors, mTOR inhibitors, antibodies(e.g., rituxan), gossyphol, genasense, polyphenol E, Chlorofusin, alltrans-retinoic acid (ATRA), bryostatin, tumor necrosis factor-relatedapoptosis-inducing ligand (TRATL), 5-aza-2′-deoxycytidine, all transretinoic acid, doxorubicin, vincristine, etoposide, gemcitabine,imatinib (Gleevec®), geldanamycin,17-N-Allylamino-17-Demethoxygeldanamycin (17-AAG), flavopiridol,LY294002, bortezomib, trastuzumab, BAY 11-7082, PKC412, PD184352,20-epi-1, 25 dihydroxyvitamin D3; 5-ethynyluracil; abiraterone;aclarubicin; acylfulvene; adecypenol; adozelesin; aldesleukin; ALL-TKantagonists; altretamine; ambamustine; amidox; amifostine;aminolevulinic acid; amrubicin; amsacrine; anagrelide; anastrozole;andrographolide; angiogenesis inhibitors; antagonist D; antagonist G;antarelix; anti-dorsalizing morphogenetic protein-1; antiandrogen,prostatic carcinoma; antiestrogen; antineoplaston; antisenseoligonucleotides; aphidicolin glycinate; apoptosis gene modulators;apoptosis regulators; apurinic acid; ara-CDP-DL-PTBA; argininedeaminase; asulacrine; atamestane; atrimustine; axinastatin 1;axinastatin 2; axinastatin 3; azasetron; azatoxin; azatyrosine; baccatinIII derivatives; balanol; batimastat; BCR/ABL antagonists;benzochlorins; benzoylstaurosporine; beta lactam derivatives;beta-alethine; betaclamycin B; betulinic acid; bFGF inhibitor;bicalutamide; bisantrene; bisaziridinylspermine; bisnafide; bistrateneA; bizelesin; breflate; bropirimine; budotitane; buthionine sulfoximine;calcipotriol; calphostin C; camptothecin derivatives; canarypox IL-2;capecitabine; carboxamide-amino-triazole; carboxyamidotriazole; CaRestM3; CARN 700; cartilage derived inhibitor; carzelesin; casein kinaseinhibitors (ICOS); castanospermine; cecropin B; cetrorelix; chlorins;chloroquinoxaline sulfonamide; cicaprost; cis-porphyrin; cladribine;clomifene analogues; clotrimazole; collismycin A; collismycin B;combretastatin A4; combretastatin analogue; conagenin; crambescidin 816;crisnatol; cryptophycin 8; cryptophycin A derivatives; curacin A;cyclopentanthraquinones; cycloplatam; cypemycin; cytarabine ocfosfate;cytolytic factor; cytostatin; dacliximab; decitabine; dehydrodidemnin B;deslorelin; dexamethasone; dexifosfamide; dexrazoxane; dexverapamil;diaziquone; didemnin B; didox; diethylnorspermine;dihydro-5-azacytidine; 9-dioxamycin; diphenyl spiromustine; docosanol;dolasetron; doxifluridine; droloxifene; dronabinol; duocarmycin SA;ebselen; ecomustine; edelfosine; edrecolomab; eflornithine; elemene;emitefur; epirubicin; epristeride; estramustine analogue; estrogenagonists; estrogen antagonists; etanidazole; etoposide phosphate;exemestane; fadrozole; fazarabine; fenretinide; filgrastim; finasteride;flavopiridol; flezelastine; fluasterone; fludarabine; fluorodaunorunicinhydrochloride; forfenimex; formestane; fostriecin; fotemustine;gadolinium texaphyrin; gallium nitrate; galocitabine; ganirelix;gelatinase inhibitors; gemcitabine; glutathione inhibitors; hepsulfam;heregulin; hexamethylene bisacetamide; hypericin; ibandronic acid;idarubicin; idoxifene; idramantone; ilmofosine; ilomastat;imidazoacridones; imiquimod; immunostimulant peptides; insulin-likegrowth factor-1 receptor inhibitor; interferon agonists; interferons;interleukins; iobenguane; iododoxorubicin; ipomeanol, 4-; iroplact;irsogladine; isobengazole; isohomohalicondrin B; itasetron;jasplakinolide; kahalalide F; lamellarin-N triacetate; lanreotide;leinamycin; lenograstim; lentinan sulfate; leptolstatin; letrozole;leukemia inhibiting factor; leukocyte alpha interferon;leuprolide+estrogen+progesterone; leuprorelin; levamisole; liarozole;linear polyamine analogue; lipophilic disaccharide peptide; lipophilicplatinum compounds; lissoclinamide 7; lobaplatin; lombricine;lometrexol; lonidamine; losoxantrone; lovastatin; loxoribine;lurtotecan; lutetium texaphyrin; lysofylline; lytic peptides;maitansine; mannostatin A; marimastat; masoprocol; maspin; matrilysininhibitors; matrix metalloproteinase inhibitors; menogaril; merbarone;meterelin; methioninase; metoclopramide; MIF inhibitor; mifepristone;miltefosine; mirimostim; mismatched double stranded RNA; mitoguazone;mitolactol; mitomycin analogues; mitonafide; mitotoxin fibroblast growthfactor-saporin; mitoxantrone; mofarotene; molgramostim; monoclonalantibody, human chorionic gonadotrophin; monophosphoryl lipidA+myobacterium cell wall sk; mopidamol; multiple drug resistance geneinhibitor; multiple tumor suppressor 1-based therapy; mustard anticanceragent; mycaperoxide B; mycobacterial cell wall extract; myriaporone;N-acetyldinaline; N-substituted benzamides; nafarelin; nagrestip;naloxone+pentazocine; napavin; naphterpin; nartograstim; nedaplatin;nemorubicin; neridronic acid; neutral endopeptidase; nilutamide;nisamycin; nitric oxide modulators; nitroxide antioxidant; nitrullyn;O6-benzylguanine; octreotide; okicenone; oligonucleotides; onapristone;ondansetron; ondansetron; oracin; oral cytokine inducer; ormaplatin;osaterone; oxaliplatin; oxaunomycin; palauamine; palmitoylrhizoxin;pamidronic acid; panaxytriol; panomifene; parabactin; pazelliptine;pegaspargase; peldesine; pentosan polysulfate sodium; pentostatin;pentrozole; perflubron; perfosfamide; perillyl alcohol; phenazinomycin;phenylacetate; phosphatase inhibitors; picibanil; pilocarpinehydrochloride; pirarubicin; piritrexim; placetin A; placetin B;plasminogen activator inhibitor; platinum complex; platinum compounds;platinum-triamine complex; porfimer sodium; porfiromycin; prednisone;propyl bis-acridone; prostaglandin J2; proteasome inhibitors; proteinA-based immune modulator; protein kinase C inhibitor; protein kinase Cinhibitors, microalgal; protein tyrosine phosphatase inhibitors; purinenucleoside phosphorylase inhibitors; purpurins; pyrazoloacridine;pyridoxylated hemoglobin polyoxyethylerie conjugate; raf antagonists;raltitrexed; ramosetron; ras farnesyl protein transferase inhibitors;ras inhibitors; ras-GAP inhibitor; retelliptine demethylated; rhenium Re186 etidronate; rhizoxin; ribozymes; RII retinamide; rogletimide;rohitukine; romurtide; roquinimex; rubiginone B1; ruboxyl; safingol;saintopin; SarCNU; sarcophytol A; sargramostim; Sdi 1 mimetics;semustine; senescence derived inhibitor 1; sense oligonucleotides;signal transduction inhibitors; signal transduction modulators; singlechain antigen-binding protein; sizofuran; sobuzoxane; sodiumborocaptate; sodium phenylacetate; solverol; somatomedin bindingprotein; sonermin; sparfosic acid; spicamycin D; spiromustine;splenopentin; spongistatin 1; squalamine; stem cell inhibitor; stem-celldivision inhibitors; stipiamide; stromelysin inhibitors; sulfinosine;superactive vasoactive intestinal peptide antagonist; suradista;suramin; swainsonine; synthetic glycosaminoglycans; tallimustine;tamoxifen methiodide; tauromustine; tazarotene; tecogalan sodium;tegafur; tellurapyrylium; telomerase inhibitors; temoporfin;temozolomide; teniposide; tetrachlorodecaoxide; tetrazomine;thaliblastine; thiocoraline; thrombopoietin; thrombopoietin mimetic;thymalfasin; thymopoietin receptor agonist; thymotrinan; thyroidstimulating hormone; tin ethyl etiopurpurin; tirapazamine; titanocenebichloride; topsentin; toremifene; totipotent stem cell factor;translation inhibitors; tretinoin; triacetyluridine; triciribine;trimetrexate; triptorelin; tropisetron; turosteride; tyrosine kinaseinhibitors; tyrphostins; UBC inhibitors; ubenimex; urogenitalsinus-derived growth inhibitory factor; urokinase receptor antagonists;vapreotide; variolin B; vector system, erythrocyte gene therapy;velaresol; veramine; verdins; verteporfin; vinorelbine; vinxaltine;vitaxin; vorozole; zanoterone; zeniplatin; zilascorb; zinostatinstimalamer, Adriamycin, Dactinomycin, Bleomycin, Vinblastine, Cisplatin,acivicin; aclarubicin; acodazole hydrochloride; acronine; adozelesin;aldesleukin; altretamine; ambomycin; ametantrone acetate;aminoglutethimide; amsacrine; anastrozole; anthramycin; asparaginase;asperlin; azacitidine; azetepa; azotomycin; batimastat; benzodepa;bicalutamide; bisantrene hydrochloride; bisnafide dimesylate; bizelesin;bleomycin sulfate; brequinar sodium; bropirimine; busulfan;cactinomycin; calusterone; caracemide; carbetimer; carboplatin;carmustine; carubicin hydrochloride; carzelesin; cedefingol;chlorambucil; cirolemycin; cladribine; crisnatol mesylate;cyclophosphamide; cytarabine; dacarbazine; daunorubicin hydrochloride;decitabine; dexormaplatin; dezaguanine; dezaguanine mesylate;diaziquone; doxorubicin; doxorubicin hydrochloride; droloxifene;droloxifene citrate; dromostanolone propionate; duazomycin; edatrexate;eflornithine hydrochloride; elsamitrucin; enloplatin; enpromate;epipropidine; epirubicin hydrochloride; erbulozole; esorubicinhydrochloride; estramustine; estramustine phosphate sodium; etanidazole;etoposide; etoposide phosphate; etoprine; fadrozole hydrochloride;fazarabine; fenretinide; floxuridine; fludarabine phosphate;fluorouracil; fluorocitabine; fosquidone; fostriecin sodium;gemcitabine; gemcitabine hydrochloride; hydroxyurea; idarubicinhydrochloride; ifosfamide; iimofosine; interleukin I1 (includingrecombinant interleukin II, or rlL.sub.2), interferon alfa-2a;interferon alfa-2b; interferon alfa-n1; interferon alfa-n3; interferonbeta-1a; interferon gamma-1b; iproplatin; irinotecan hydrochloride;lanreotide acetate; letrozole; leuprolide acetate; liarozolehydrochloride; lometrexol sodium; lomustine; losoxantrone hydrochloride;masoprocol; maytansine; mechlorethamine hydrochloride; megestrolacetate; melengestrol acetate; melphalan; menogaril; mercaptopurine;methotrexate; methotrexate sodium; metoprine; meturedepa; mitindomide;mitocarcin; mitocromin; mitogillin; mitomalcin; mitomycin; mitosper;mitotane; mitoxantrone hydrochloride; mycophenolic acid; nocodazoie;nogalamycin; ormaplatin; oxisuran; pegaspargase; peliomycin;pentamustine; peplomycin sulfate; perfosfamide; pipobroman; piposulfan;piroxantrone hydrochloride; plicamycin; plomestane; porfimer sodium;porfiromycin; prednimustine; procarbazine hydrochloride; puromycin;puromycin hydrochloride; pyrazofurin; riboprine; rogletimide; safingol;safingol hydrochloride; semustine; simtrazene; sparfosate sodium;sparsomycin; spirogermanium hydrochloride; spiromustine; spiroplatin;streptonigrin; streptozocin; sulofenur; talisomycin; tecogalan sodium;tegafur; teloxantrone hydrochloride; temoporfin; teniposide; teroxirone;testolactone; thiamiprine; thioguanine; thiotepa; tiazofurin;tirapazamine; toremifene citrate; trestolone acetate; triciribinephosphate; trimetrexate; trimetrexate glucuronate; triptorelin;tubulozole hydrochloride; uracil mustard; uredepa; vapreotide;verteporfin; vinblastine sulfate; vincristine sulfate; vindesine;vindesine sulfate; vinepidine sulfate; vinglycinate sulfate;vinleurosine sulfate; vinorelbine tartrate; vinrosidine sulfate;vinzolidine sulfate; vorozole; zeniplatin; zinostatin; zorubicinhydrochloride, agents that arrest cells in the G2-M phases and/ormodulate the formation or stability of microtubules, (e.g. Taxol™ (i.e.paclitaxel), Taxotere™, compounds comprising the taxane skeleton,Erbulozole (i.e. R-55104), Dolastatin 10 (i.e. DLS-10 and NSC-376128),Mivobulin isethionate (i.e. as CI-980), Vincristine, NSC-639829,Discodermolide (i.e. as NVP-XX-A-296), ABT-751 (Abbott, i.e. E-7010),Altorhyrtins (e.g. Altorhyrtin A and Altorhyrtin C), Spongistatins (e.g.Spongistatin 1, Spongistatin 2, Spongistatin 3, Spongistatin 4,Spongistatin 5, Spongistatin 6, Spongistatin 7, Spongistatin 8, andSpongistatin 9), Cemadotin hydrochloride (i.e. LU-103793 andNSC-D-669356), Epothilones (e.g. Epothilone A, Epothilone B, EpothiloneC (i.e. desoxyepothilone A or dEpoA), Epothilone D (i.e. KOS-862, dEpoB,and desoxyepothilone B), Epothilone E, Epothilone F, Epothilone BN-oxide, Epothilone A N-oxide, 16-aza-epothilone B, 21-aminoepothilone B(i.e. BMS-310705), 21-hydroxyepothilone D (i.e. Desoxyepothilone F anddEpoF), 26-fluoroepothilone, Auristatin PE (i.e. NSC-654663), Soblidotin(i.e. TZT-1027), LS-4559-P (Pharmacia, i.e. LS-4577), LS-4578(Pharmacia, i.e. LS-477-P), LS-4477 (Pharmacia), LS-4559 (Pharmacia),RPR-112378 (Aventis), Vincristine sulfate, DZ-3358 (Daiichi), FR-182877(Fujisawa, i.e. WS-9885B), GS-164 (Takeda), GS-198 (Takeda), KAR-2(Hungarian Academy of Sciences), BSF-223651 (BASF, i.e. ILX-651 andLU-223651), SAH-49960 (Lilly/Novartis), SDZ-268970 (Lilly/Novartis),AM-97 (Armad/Kyowa Hakko), AM-132 (Armad), AM-138 (Armad/Kyowa Hakko),IDN-5005 (Indena), Cryptophycin 52 (i.e. LY-355703), AC-7739 (Ajinomoto,i.e. AVE-8063A and CS-39.HCl), AC-7700 (Ajinomoto, i.e. AVE-8062,AVE-8062A, CS-39-L-Ser.HCl, and RPR-258062A), Vitilevuamide, TubulysinA, Canadensol, Centaureidin (i.e. NSC-106969), T-138067 (Tularik, i.e.T-67, TL-138067 and TI-138067), COBRA-1 (Parker Hughes Institute, i.e.DDE-261 and WHI-261), H10 (Kansas State University), H16 (Kansas StateUniversity), Oncocidin A1 (i.e. BTO-956 and DIME), DDE-313 (ParkerHughes Institute), Fijianolide B, Laulimalide, SPA-2 (Parker HughesInstitute), SPA-1 (Parker Hughes Institute, i.e. SPIKET-P), 3-IAABU(Cytoskeleton/Mt. Sinai School of Medicine, i.e. MF-569), Narcosine(also known as NSC-5366), Nascapine, D-24851 (Asta Medica), A-105972(Abbott), Hemiasterlin, 3-BAABU (Cytoskeleton/Mt. Sinai School ofMedicine, i.e. MF-191), TMPN (Arizona State University), Vanadoceneacetylacetonate, T-138026 (Tularik), Monsatrol, lnanocine (i.e.NSC-698666), 3-IAABE (Cytoskeleton/Mt. Sinai School of Medicine),A-204197 (Abbott), T-607 (Tuiarik, i.e. T-900607), RPR-115781 (Aventis),Eleutherobins (such as Desmethyleleutherobin, Desaetyleleutherobin,lsoeleutherobin A, and Z-Eleutherobin), Caribaeoside, Caribaeolin,Halichondrin B, D-64131 (Asta Medica), D-68144 (Asta Medica),Diazonamide A, A-293620 (Abbott), NPI-2350 (Nereus), Taccalonolide A,TUB-245 (Aventis), A-259754 (Abbott), Diozostatin, (−)-Phenylahistin(i.e. NSCL-96F037), D-68838 (Asta Medica), D-68836 (Asta Medica),Myoseverin B, D-43411 (Zentaris, i.e. D-81862), A-289099 (Abbott),A-318315 (Abbott), HTI-286 (i.e. SPA-110, trifluoroacetate salt)(Wyeth), D-82317 (Zentaris), D-82318 (Zentaris), SC-12983 (NCI),Resverastatin phosphate sodium, BPR-OY-007 (National Health ResearchInstitutes), and SSR-250411 (Sanofi)), steroids (e.g., dexamethasone),finasteride, aromatase inhibitors, gonadotropin-releasing hormoneagonists (GnRH) such as goserelin or leuprolide, adrenocorticosteroids(e.g., prednisone), progestins (e.g., hydroxyprogesterone caproate,megestrol acetate, medroxyprogesterone acetate), estrogens (e.g.,diethlystilbestrol, ethinyl estradiol), antiestrogen (e.g., tamoxifen),androgens (e.g., testosterone propionate, fluoxymesterone), antiandrogen(e.g., flutamide), immunostimulants (e.g., Bacillus Calmette-Guerin(BCG), levamisole, interleukin-2, alpha-interferon, etc.), monoclonalantibodies (e.g., anti-CD20, anti-HER2, anti-CD52, anti-HLA-DR, andanti-VEGF monoclonal antibodies), immunotoxins (e.g., anti-CD33monoclonal antibody-calicheamicin conjugate, anti-CD22 monoclonalantibody-pseudomonas exotoxin conjugate, etc.), radioimmunotherapy(e.g., anti-CD20 monoclonal antibody conjugated to 111In, 90Y, or 131I,etc.), triptolide, homoharringtonine, dactinomycin, doxorubicin,epirubicin, topotecan, itraconazole, vindesine, cerivastatin,vincristine, deoxyadenosine, sertraline, pitavastatin, irinotecan,clofazimine, 5-nonyloxytryptamine, vemurafenib, dabrafenib, erlotinib,gefitinib, EGFR inhibitors, epidermal growth factor receptor(EGFR)-targeted therapy or therapeutic (e.g. gefitinib (Iressa™)erlotinib (Tarceva™), cetuximab (Erbitux™), lapatinib (Tykerb™),panitumumab (Vectibix™), vandetanib (Caprelsa™), afatinib/BIBW2992,CI-1033/canertinib, neratinib/HKI-272, CP-724714, TAK-285, AST-1306,ARRY334543, ARRY-380, AG-1478, dacomitinib/PF299804, OSI-420/desmethylerlotinib, AZD8931, AEE788, pelitinib/EKB-569, CUDC-101, WZ8040, WZ4002,WZ3146, AG-490, XL647, PD153035, BMS-599626), sorafenib, imatinib,sunitinib, dasatinib, or the like.

As used herein, the term “administering” means oral administration,administration as a suppository, topical contact, intravenous,intraperitoneal, intramuscular, intralesional, intrathecal, intranasalor subcutaneous administration, or the implantation of a slow-releasedevice, e.g., a mini-osmotic pump, to a subject. Administration is byany route, including parenteral and transmucosal (e.g., buccal,sublingual, palatal, gingival, nasal, vaginal, rectal, or transdermal).Parenteral administration includes, e.g., intravenous, intramuscular,intra-arteriole, intradermal, subcutaneous, intraperitoneal,intraventricular, and intracranial. Other modes of delivery include, butare not limited to, the use of liposomal formulations, intravenousinfusion, transdermal patches, etc. By “co-administer” it is meant thata composition described herein is administered at the same time, justprior to, or just after the administration of one or more additionaltherapies, for example cancer therapies such as chemotherapy, hormonaltherapy, radiotherapy, or immunotherapy. The compounds of the invention(e.g., antibody, bispecific antibody or chimeric antigen receptor) canbe administered alone or can be coadministered to the patient.Coadministration is meant to include simultaneous or sequentialadministration of the compounds individually or in combination (morethan one compound). Thus, the preparations can also be combined, whendesired, with other active substances (e.g. to reduce metabolicdegradation). The compositions of the present invention can be deliveredby transdermally, by a topical route, formulated as applicator sticks,solutions, suspensions, emulsions, gels, creams, ointments, pastes,jellies, paints, powders, and aerosols.

The compositions of the present invention may additionally includecomponents to provide sustained release and/or comfort. Such componentsinclude high molecular weight, anionic mucomimetic polymers, gellingpolysaccharides and finely-divided drug carrier substrates. Thesecomponents are discussed in greater detail in U.S. Pat. Nos. 4,911,920;5,403,841; 5,212,162; and 4,861,760. The entire contents of thesepatents are incorporated herein by reference in their entirety for allpurposes. The compositions of the present invention can also bedelivered as microspheres for slow release in the body. For example,microspheres can be administered via intradermal injection ofdrug-containing microspheres, which slowly release subcutaneously (seeRao, J. Biomater Sci. Polym. Ed. 7:623-645, 1995; as biodegradable andinjectable gel formulations (see, e.g., Gao Pharm. Res. 12:857-863,1995); or, as microspheres for oral administration (see, e.g., Eyles, J.Pharm. Pharmacol. 49:669-674, 1997). In embodiments, the formulations ofthe compositions of the present invention can be delivered by the use ofliposomes which fuse with the cellular membrane or are endocytosed,i.e., by employing receptor ligands attached to the liposome, that bindto surface membrane protein receptors of the cell resulting inendocytosis. By using liposomes, particularly where the liposome surfacecarries receptor ligands specific for target cells, or are otherwisepreferentially directed to a specific organ, one can focus the deliveryof the compositions of the present invention into the target cells invivo. (See, e.g., Al-Muhammed, J. Microencapsul. 13:293-306, 1996;Chonn, Curr. Opin. Biotechnol. 6:698-708, 1995; Ostro, Am. J. Hosp.Pharm. 46:1576-1587, 1989). The compositions of the present inventioncan also be delivered as nanoparticles.

As used herein, the term “pharmaceutically acceptable” is usedsynonymously with “physiologically acceptable” and “pharmacologicallyacceptable”. A pharmaceutical composition will generally comprise agentsfor buffering and preservation in storage, and can include buffers andcarriers for appropriate delivery, depending on the route ofadministration.

“Pharmaceutically acceptable excipient” and “pharmaceutically acceptablecarrier” refer to a substance that aids the administration of an activeagent to and absorption by a subject and can be included in thecompositions of the present invention without causing a significantadverse toxicological effect on the patient. Non-limiting examples ofpharmaceutically acceptable excipients include water, NaCl, normalsaline solutions, lactated Ringer's, normal sucrose, normal glucose,binders, fillers, disintegrants, lubricants, coatings, sweeteners,flavors, salt solutions (such as Ringer's solution), alcohols, oils,gelatins, carbohydrates such as lactose, amylose or starch, fatty acidesters, hydroxymethycellulose, polyvinyl pyrrolidine, and colors, andthe like. Such preparations can be sterilized and, if desired, mixedwith auxiliary agents such as lubricants, preservatives, stabilizers,wetting agents, emulsifiers, salts for influencing osmotic pressure,buffers, coloring, and/or aromatic substances and the like that do notdeleteriously react with the compounds of the invention. One of skill inthe art will recognize that other pharmaceutical excipients are usefulin the present invention.

The term “pharmaceutically acceptable salt” refers to salts derived froma variety of organic and inorganic counter ions well known in the artand include, by way of example only, sodium, potassium, calcium,magnesium, ammonium, tetraalkylammonium, and the like; and when themolecule contains a basic functionality, salts of organic or inorganicacids, such as hydrochloride, hydrobromide, tartrate, mesylate, acetate,maleate, oxalate and the like.

The term “preparation” is intended to include the formulation of theactive compound with encapsulating material as a carrier providing acapsule in which the active component with or without other carriers, issurrounded by a carrier, which is thus in association with it.Similarly, cachets and lozenges are included. Tablets, powders,capsules, pills, cachets, and lozenges can be used as solid dosage formssuitable for oral administration.

The pharmaceutical preparation is optionally in unit dosage form. Insuch form the preparation is subdivided into unit doses containingappropriate quantities of the active component. The unit dosage form canbe a packaged preparation, the package containing discrete quantities ofpreparation, such as packeted tablets, capsules, and powders in vials orampoules. Also, the unit dosage form can be a capsule, tablet, cachet,or lozenge itself, or it can be the appropriate number of any of thesein packaged form. The unit dosage form can be of a frozen dispersion.

Antibody Compositions

Provided herein are, inter alia, antibodies capable of bindingInterleukin-1 receptor accessory protein (IL1RAP). The antibodiesprovided herein include novel light chain and heavy chain sequences andbind IL1RAP with high efficiency and specificity, thereby blocking IL-1signaling. Blocking of IL-1 signaling results in the inhibition ofproliferation of IL1RAP-expressing cells. Further, through therecruitment of effector cells, the anti-ILRAP1 antibodies providedherein are able to induce cell killing of IL1RAP-expressing cells.IL1RAP is expressed on a variety of cell types, for example, oncandidate leukemic stem cells acute myeloid leukemia (AML) patients, butnot on normal hematopoietic stem cells. Thus, the anti-IL1RAP antibodiesprovided herein are, inter alia, useful for the treatment ofIL1RAP-expressing cancers such as AML.

In an aspect is provided an anti-interleukin-1 receptor accessoryprotein (IL1RAP) antibody including a light chain variable domain and aheavy chain variable domain, wherein the light chain variable domainincludes: a CDR L1 as set forth in SEQ ID NO:1, a CDR L2 as set forth inSEQ ID NO:2 and a CDR L3 as set forth in SEQ ID NO:3; and wherein theheavy chain variable domain includes: a CDR H1 as set forth in SEQ IDNO:4, a CDR H2 as set forth in SEQ ID NO:5, and a CDR H3 as set forth inSEQ ID NO: 6.

As described above, a “light chain variable (VL) domain” as providedherein refers to the variable region of the light chain of an antibody,an antibody variant or fragment thereof. Likewise, the “heavy chainvariable (VH) domain” as provided herein refers to the variable regionof the heavy chain of an antibody, an antibody variant or fragmentthereof. The light chain variable domain and the heavy chain variabledomain together form the paratope, which binds an antigen (epitope). Theparatope or antigen-binding site is formed at the N-terminus of anantibody, an antibody variant or fragment thereof. In embodiments, thelight chain variable (VL) domain includes CDR L1, CDR L2, CDR L3 and FRL1, FR L2, FR L3 and FR L4 (framework regions) of an antibody lightchain. In embodiments, the heavy chain variable (VH) domain includes CDRH1, CDR H2, CDR H3 and FR H1, FR H2, FR H3 and FR H4 (framework regions)of an antibody heavy chain. In embodiments, the light chain variable(VL) domain and a light chain constant (CL) domain form part of anantibody light chain. In embodiments, the heavy chain variable (VH)domain and a heavy chain constant (CH1) domain form part of an antibodyheavy chain. In embodiments, the heavy chain variable (VH) domain andone or more heavy chain constant (CH1, CH2, or CH3) domains form part ofan antibody heavy chain. Thus, in embodiments, the light chain variable(VL) domain forms part of an antibody. In embodiments, the heavy chainvariable (VH) domain forms part of an antibody. In embodiments, thelight chain variable (VL) domain forms part of a therapeutic antibody.In embodiments, the heavy chain variable (VH) domain forms part of atherapeutic antibody. In embodiments, the light chain variable (VL)domain forms part of a human antibody. In embodiments, the heavy chainvariable (VH) domain forms part of a human antibody. In embodiments, thelight chain variable (VL) domain forms part of a humanized antibody. Inembodiments, the heavy chain variable (VH) domain forms part of ahumanized antibody. In embodiments, the light chain variable (VL) domainforms part of a chimeric antibody. In embodiments, the heavy chainvariable (VH) domain forms part of a chimeric antibody. In embodiments,the light chain variable (VL) domain forms part of an antibody fragment.In embodiments, the heavy chain variable (VH) domain forms part of anantibody fragment. In embodiments, the light chain variable (VL) domainforms part of an antibody variant. In embodiments, the heavy chainvariable (VH) domain forms part of an antibody variant. In embodiments,the light chain variable (VL) domain forms part of a Fab. Inembodiments, the heavy chain variable (VH) domain forms part of a Fab.In embodiments, the light chain variable (VL) domain forms part of ascFv. In embodiments, the heavy chain variable (VH) domain forms part ofa scFv.

In embodiments, the light chain variable domain includes the sequence ofSEQ ID NO:7. In embodiments, the light chain variable domain is thesequence of SEQ ID NO:7. In embodiments, the heavy chain variable domainincludes the sequence of SEQ ID NO:8. In embodiments, the heavy chainvariable domain is the sequence of SEQ ID NO:8. In embodiments, lightchain variable domain includes a FR L1 as set forth in SEQ ID NO:9, a FRL2 as set forth in SEQ ID NO:10, FR L3 as set forth in SEQ ID NO:11 anda FR L4 as set forth in SEQ ID NO:12. In embodiments, the heavy chainvariable domain includes a FR H1 as set forth in SEQ ID NO:13, a FR H2as set forth in SEQ ID NO:14, FR H3 as set forth in SEQ ID NO:15 and aFR H4 as set forth in SEQ ID NO:16.

In one embodiment, the antibody includes (i) a light chain variabledomain including a CDR L1 as set forth in SEQ ID NO: 1, a CDR L2 as setforth in SEQ ID NO:2; a CDR L3 as set forth in SEQ ID NO:3; a FR L1 asset forth in SEQ ID NO:9, a FR L2 as set forth in SEQ ID NO:10, FR L3 asset forth in SEQ ID NO:11 and a FR L4 as set forth in SEQ ID NO:12; and(ii) a heavy chain variable domain including a CDR H1 as set forth inSEQ ID NO:4, a CDR H2 as set forth in SEQ ID NO:5, a CDR H3 as set forthin SEQ ID NO:6; a FR H1 as set forth in SEQ ID NO:13, a FR H2 as setforth in SEQ ID NO:14, FR H3 as set forth in SEQ ID NO:15 and a FR H4 asset forth in SEQ ID NO:16.

In one embodiment, the antibody includes a light chain variable domainof SEQ ID NO:7 and a heavy chain variable domain of SEQ ID NO:8.

In embodiments, the antibody is an IgG. In embodiments, the antibody isa human IgG. In embodiments, the antibody is an IgG1. In embodiments,the antibody is a human IgG1.

In embodiments, the antibody is a Fab′ fragment. In embodiments, theantibody forms part of a Fab′ fragment. In embodiments, the antibody isa single chain antibody (scFv). In embodiments, the light chain variabledomain and the heavy chain variable domain form part of an scFv. Inembodiments, the scFv includes the sequence of SEQ ID NO:17. Inembodiments, the scFv is the sequence of SEQ ID NO:17.

The ability of an antibody to bind a specific epitope (e.g., IL1RAP) canbe described by the equilibrium dissociation constant (K_(D)). Theequilibrium dissociation constant (K_(D)) as defined herein is the ratioof the dissociation rate (K-off) and the association rate (K-on) of anantibody to IL1RAP. It is described by the following formula:K_(D)=K-off/K-on. In embodiments, the antibody is capable of bindingIL1RAP with an equilibrium dissociation constant (K_(D)) of about 21 nM.In embodiments, the antibody is capable of binding IL1RAP with anequilibrium dissociation constant (K_(D)) of 21 nM. In embodiments, theantibody is capable of binding IL1RAP with an equilibrium dissociationconstant (K_(D)) of about 80.6 nM. In embodiments, the antibody iscapable of binding IL1RAP with an equilibrium dissociation constant(K_(D)) of 80.6 nM.

In embodiments, the antibody is capable of binding IL1RAP with anequilibrium dissociation constant (K_(D)) from 20 to 25 nM. Inembodiments, the antibody is capable of binding IL1RAP with anequilibrium dissociation constant (K_(D)) from 21 to 25 nM. Inembodiments, the antibody is capable of binding IL1RAP with anequilibrium dissociation constant (K_(D)) from 22 to 25 nM. Inembodiments, the antibody is capable of binding IL1RAP with anequilibrium dissociation constant (K_(D)) from 23 to 25 nM. Inembodiments, the antibody is capable of binding IL1RAP with anequilibrium dissociation constant (K_(D)) from 24 to 25 nM.

In embodiments, the antibody is capable of binding IL1RAP with anequilibrium dissociation constant (K_(D)) from 75 to 85 nM. Inembodiments, the antibody is capable of binding IL1RAP with anequilibrium dissociation constant (K_(D)) from 76 to 85 nM. Inembodiments, the antibody is capable of binding IL1RAP with anequilibrium dissociation constant (K_(D)) from 77 to 85 nM. Inembodiments, the antibody is capable of binding IL1RAP with anequilibrium dissociation constant (K_(D)) from 78 to 85 nM. Inembodiments, the antibody is capable of binding IL1RAP with anequilibrium dissociation constant (K_(D)) from 79 to 85 nM. Inembodiments, the antibody is capable of binding IL1RAP with anequilibrium dissociation constant (K_(D)) from 80 to 85 nM. Inembodiments, the antibody is capable of binding IL1RAP with anequilibrium dissociation constant (K_(D)) from 81 to 85 nM. Inembodiments, the antibody is capable of binding IL1RAP with anequilibrium dissociation constant (K_(D)) from 82 to 85 nM. Inembodiments, the antibody is capable of binding IL1RAP with anequilibrium dissociation constant (K_(D)) from 83 to 85 nM. Inembodiments, the antibody is capable of binding IL1RAP with anequilibrium dissociation constant (K_(D)) from 84 to 85 nM.

In embodiments, the antibody is capable of binding IL1RAP with anequilibrium dissociation constant (K_(D)) from about 20 to about 85 nM.In embodiments, the antibody is capable of binding IL1RAP with anequilibrium dissociation constant (K_(D)) from about 25 to about 85 nM.In embodiments, the antibody is capable of binding IL1RAP with anequilibrium dissociation constant (K_(D)) from about 30 to about 85 nM.In embodiments, the antibody is capable of binding IL1RAP with anequilibrium dissociation constant (K_(D)) from about 35 to about 85 nM.In embodiments, the antibody is capable of binding IL1RAP with anequilibrium dissociation constant (K_(D)) from about 40 to about 85 nM.In embodiments, the antibody is capable of binding IL1RAP with anequilibrium dissociation constant (K_(D)) from about 45 to about 85 nM.In embodiments, the antibody is capable of binding IL1RAP with anequilibrium dissociation constant (K_(D)) from about 50 to about 85 nM.In embodiments, the antibody is capable of binding IL1RAP with anequilibrium dissociation constant (K_(D)) from about 55 to about 85 nM.In embodiments, the antibody is capable of binding IL1RAP with anequilibrium dissociation constant (K_(D)) from about 60 to about 85 nM.In embodiments, the antibody is capable of binding IL1RAP with anequilibrium dissociation constant (K_(D)) from about 65 to about 85 nM.In embodiments, the antibody is capable of binding IL1RAP with anequilibrium dissociation constant (K_(D)) from about 70 to about 85 nM.In embodiments, the antibody is capable of binding IL1RAP with anequilibrium dissociation constant (K_(D)) from about 75 to about 85 nM.In embodiments, the antibody is capable of binding IL1RAP with anequilibrium dissociation constant (K_(D)) from about 80 to about 85 nM.

In embodiments, the antibody is capable of binding IL1RAP with anequilibrium dissociation constant (K_(D)) from about 20 to about 80 nM.In embodiments, the antibody is capable of binding IL1RAP with anequilibrium dissociation constant (K_(D)) from about 25 to about 80 nM.In embodiments, the antibody is capable of binding IL1RAP with anequilibrium dissociation constant (K_(D)) from about 30 to about 80 nM.In embodiments, the antibody is capable of binding IL1RAP with anequilibrium dissociation constant (K_(D)) from about 35 to about 80 nM.In embodiments, the antibody is capable of binding IL1RAP with anequilibrium dissociation constant (K_(D)) from about 40 to about 80 nM.In embodiments, the antibody is capable of binding IL1RAP with anequilibrium dissociation constant (K_(D)) from about 45 to about 80 nM.In embodiments, the antibody is capable of binding IL1RAP with anequilibrium dissociation constant (K_(D)) from about 50 to about 80 nM.In embodiments, the antibody is capable of binding IL1RAP with anequilibrium dissociation constant (K_(D)) from about 55 to about 80 nM.In embodiments, the antibody is capable of binding IL1RAP with anequilibrium dissociation constant (K_(D)) from about 60 to about 80 nM.In embodiments, the antibody is capable of binding IL1RAP with anequilibrium dissociation constant (K_(D)) from about 65 to about 80 nM.In embodiments, the antibody is capable of binding IL1RAP with anequilibrium dissociation constant (K_(D)) from about 70 to about 80 nM.In embodiments, the antibody is capable of binding IL1RAP with anequilibrium dissociation constant (K_(D)) from about 75 to about 80 nM.

In embodiments, the antibody is capable of binding IL1RAP with anequilibrium dissociation constant (K_(D)) from about 20 to about 70 nM.In embodiments, the antibody is capable of binding IL1RAP with anequilibrium dissociation constant (K_(D)) from about 25 to about 70 nM.In embodiments, the antibody is capable of binding IL1RAP with anequilibrium dissociation constant (K_(D)) from about 30 to about 70 nM.In embodiments, the antibody is capable of binding IL1RAP with anequilibrium dissociation constant (K_(D)) from about 35 to about 70 nM.In embodiments, the antibody is capable of binding IL1RAP with anequilibrium dissociation constant (K_(D)) from about 40 to about 70 nM.In embodiments, the antibody is capable of binding IL1RAP with anequilibrium dissociation constant (K_(D)) from about 45 to about 70 nM.In embodiments, the antibody is capable of binding IL1RAP with anequilibrium dissociation constant (K_(D)) from about 50 to about 70 nM.In embodiments, the antibody is capable of binding IL1RAP with anequilibrium dissociation constant (K_(D)) from about 55 to about 70 nM.In embodiments, the antibody is capable of binding IL1RAP with anequilibrium dissociation constant (K_(D)) from about 60 to about 70 nM.In embodiments, the antibody is capable of binding IL1RAP with anequilibrium dissociation constant (K_(D)) from about 65 to about 70 nM.

In embodiments, the antibody is capable of binding IL1RAP with anequilibrium dissociation constant (K_(D)) from about 20 to about 60 nM.In embodiments, the antibody is capable of binding IL1RAP with anequilibrium dissociation constant (K_(D)) from about 25 to about 60 nM.In embodiments, the antibody is capable of binding IL1RAP with anequilibrium dissociation constant (K_(D)) from about 30 to about 60 nM.In embodiments, the antibody is capable of binding IL1RAP with anequilibrium dissociation constant (K_(D)) from about 35 to about 60 nM.In embodiments, the antibody is capable of binding IL1RAP with anequilibrium dissociation constant (K_(D)) from about 40 to about 60 nM.In embodiments, the antibody is capable of binding IL1RAP with anequilibrium dissociation constant (K_(D)) from about 45 to about 60 nM.In embodiments, the antibody is capable of binding IL1RAP with anequilibrium dissociation constant (K_(D)) from about 50 to about 60 nM.In embodiments, the antibody is capable of binding IL1RAP with anequilibrium dissociation constant (K_(D)) from about 55 to about 60 nM.

In embodiments, the antibody is capable of binding IL1RAP with anequilibrium dissociation constant (K_(D)) from about 20 to about 50 nM.In embodiments, the antibody is capable of binding IL1RAP with anequilibrium dissociation constant (K_(D)) from about 25 to about 50 nM.In embodiments, the antibody is capable of binding IL1RAP with anequilibrium dissociation constant (K_(D)) from about 30 to about 50 nM.In embodiments, the antibody is capable of binding IL1RAP with anequilibrium dissociation constant (K_(D)) from about 35 to about 50 nM.In embodiments, the antibody is capable of binding IL1RAP with anequilibrium dissociation constant (K_(D)) from about 40 to about 50 nM.In embodiments, the antibody is capable of binding IL1RAP with anequilibrium dissociation constant (K_(D)) from about 45 to about 50 nM.

In embodiments, the antibody is capable of binding IL1RAP with anequilibrium dissociation constant (K_(D)) from about 20 to about 40 nM.In embodiments, the antibody is capable of binding IL1RAP with anequilibrium dissociation constant (K_(D)) from about 25 to about 40 nM.In embodiments, the antibody is capable of binding IL1RAP with anequilibrium dissociation constant (K_(D)) from about 30 to about 40 nM.In embodiments, the antibody is capable of binding IL1RAP with anequilibrium dissociation constant (K_(D)) from about 35 to about 40 nM.

In embodiments, the antibody is capable of binding IL1RAP with anequilibrium dissociation constant (K_(D)) from about 20 to about 30 nM.In embodiments, the antibody is capable of binding IL1RAP with anequilibrium dissociation constant (K_(D)) from about 25 to about 30 nM.

In embodiments, the antibody has an EC₅₀ from about 1 ng/ml to about 20ng/ml. In embodiments, the antibody has an EC₅₀ from about 2 ng/ml toabout 20 ng/ml. In embodiments, the antibody has an EC₅₀ from about 3ng/ml to about 20 ng/ml. In embodiments, the antibody has an EC₅₀ fromabout 4 ng/ml to about 20 ng/ml. In embodiments, the antibody has anEC₅₀ from about 5 ng/ml to about 20 ng/ml. In embodiments, the antibodyhas an EC₅₀ from about 6 ng/ml to about 20 ng/ml. In embodiments, theantibody has an EC₅₀ from about 7 ng/ml to about 20 ng/ml. Inembodiments, the antibody has an EC₅₀ from about 8 ng/ml to about 20ng/ml. In embodiments, the antibody has an EC₅₀ from about 9 ng/ml toabout 20 ng/ml. In embodiments, the antibody has an EC₅₀ from about 10ng/ml to about 20 ng/ml.

In embodiments, the antibody has an EC₅₀ from about 11 ng/ml to about 20ng/ml. In embodiments, the antibody has an EC₅₀ from about 12 ng/ml toabout 20 ng/ml. In embodiments, the antibody has an EC₅₀ from about 13ng/ml to about 20 ng/ml. In embodiments, the antibody has an EC₅₀ fromabout 14 ng/ml to about 20 ng/ml. In embodiments, the antibody has anEC₅₀ from about 15 ng/ml to about 20 ng/ml. In embodiments, the antibodyhas an EC₅₀ from about 16 ng/ml to about 20 ng/ml. In embodiments, theantibody has an EC₅₀ from about 17 ng/ml to about 20 ng/ml. Inembodiments, the antibody has an EC₅₀ from about 18 ng/ml to about 20ng/ml. In embodiments, the antibody has an EC₅₀ from about 19 ng/ml toabout 20 ng/ml.

In embodiments, the antibody has an EC₅₀ from about 1 to about 19 ng/ml.In embodiments, the antibody has an EC₅₀ from about 1 ng/ml to about 18ng/ml. In embodiments, the antibody has an EC₅₀ from about 1 ng/ml toabout 17 ng/ml. In embodiments, the antibody has an EC₅₀ from about 1ng/ml to about 16 ng/ml. In embodiments, the antibody has an EC₅₀ fromabout 1 ng/ml to about 15 ng/ml. In embodiments, the antibody has anEC₅₀ from about 1 ng/ml to about 14 ng/ml. In embodiments, the antibodyhas an EC₅₀ from about 1 ng/ml to about 13 ng/ml. In embodiments, theantibody has an EC₅₀ from about 1 ng/ml to about 12 ng/ml. Inembodiments, the antibody has an EC₅₀ from about 1 ng/ml to about 11ng/ml.

In embodiments, the antibody has an EC₅₀ from about 1 ng/ml to about 10ng/ml. In embodiments, the antibody has an EC₅₀ from about 1 ng/ml toabout 9 ng/ml. In embodiments, the antibody has an EC₅₀ from about 1ng/ml to about 8 ng/ml. In embodiments, the antibody has an EC₅₀ fromabout 1 ng/ml to about 7 ng/ml. In embodiments, the antibody has an EC₅₀from about 1 ng/ml to about 6 ng/ml. In embodiments, the antibody has anEC₅₀ from about 1 ng/ml to about 5 ng/ml. In embodiments, the antibodyhas an EC₅₀ from about 1 ng/ml to about 4 ng/ml. In embodiments, theantibody has an EC₅₀ from about 1 ng/ml to about 3 ng/ml. Inembodiments, the antibody has an EC₅₀ from about 1 ng/ml to about 2ng/ml.

In embodiments, the antibody has an EC₅₀ of about 1 ng/ml, 2 ng/ml, 4ng/ml, 5 ng/ml, 6 ng/ml, 7 ng/ml, 8 ng/ml, 9 ng/ml, 10 ng/ml, 11 ng/ml,12 ng/ml, 13 ng/ml, 14 ng/ml, 15 ng/ml, 16 ng/ml, 17 ng/ml, 18 ng/ml, 19ng/ml or 20 ng/ml. In embodiments, the antibody has an EC₅₀ of 1 ng/ml,2 ng/ml, 4 ng/ml, 5 ng/ml, 6 ng/ml, 7 ng/ml, 8 ng/ml, 9 ng/ml, 10 ng/ml,11 ng/ml, 12 ng/ml, 13 ng/ml, 14 ng/ml, 15 ng/ml, 16 ng/ml, 17 ng/ml, 18ng/ml, 19 ng/ml or 20 ng/ml.

In embodiments, the antibody has an EC₅₀ from about 4 ng/ml to about 7ng/ml. In embodiments, the antibody has an EC₅₀ from about 4.5 ng/ml toabout 7 ng/ml. In embodiments, the antibody has an EC₅₀ from about 5ng/ml to about 7 ng/ml. In embodiments, the antibody has an EC₅₀ fromabout 5.5 ng/ml to about 7 ng/ml. In embodiments, the antibody has anEC₅₀ from about 6 ng/ml to about 7 ng/ml. In embodiments, the antibodyhas an EC₅₀ from about 6.5 ng/ml to about 7 ng/ml.

In embodiments, the antibody has an EC₅₀ from 4 ng/ml to 7 ng/ml. Inembodiments, the antibody has an EC₅₀ from 4.5 ng/ml to 7 ng/ml. Inembodiments, the antibody has an EC₅₀ from 5 ng/ml to 7 ng/ml. Inembodiments, the antibody has an EC₅₀ from 5.5 ng/ml to 7 ng/ml. Inembodiments, the antibody has an EC₅₀ from 6 ng/ml to 7 ng/ml. Inembodiments, the antibody has an EC₅₀ from 6.5 ng/ml to 7 ng/ml.

In embodiments, the antibody has an EC₅₀ of about 4.9 ng/ml. Inembodiments, the antibody has an EC₅₀ of 4.9 ng/ml. In embodiments, theantibody has an EC₅₀ of about 6.8 ng/ml. In embodiments, the antibodyhas an EC₅₀ of 6.8 ng/ml. In embodiments, the antibody has an EC₅₀ ofabout 6.4 ng/ml. In embodiments, the antibody has an EC₅₀ of 6.4 ng/ml.In embodiments, the antibody has an EC₅₀ of about 5.4 ng/ml. Inembodiments, the antibody has an EC₅₀ of 5.4 ng/ml.

In embodiments, the antibody is bound to an IL1RAP. In embodiments, theIL1RAP is a human IL1RAP. In embodiments, the IL1RAP forms part of acell. In embodiments, the IL1RAP is expressed on the surface of thecell. In embodiments, the cell is a cancer cell. In embodiments, thecancer cell is a leukemia stem cell (LSC). In embodiments, the cancercell is an acute myeloid leukemia (AML) cell. In embodiments, the cancercell is a chronic myeloid leukemia (CML) cell. In embodiments, thecancer cell is a lung cancer cell. In embodiments, the cancer cell is anon-small cell lung cancer (NSCLC) cell. In embodiments, the cancer cellis a pancreatic cancer cell. In embodiments, the cancer cell is amelanoma cell. In embodiments, the cancer cell is a breast cancer cell.In embodiments, the cancer cell is a colon cancer cell.

In embodiments, the anti-interleukin-1 receptor accessory protein(IL1RAP) antibody has the sequence of SEQ ID NO: 17.

Recombinant Protein Compositions

As described above, the light chain variable (VL) domain and the heavychain variable (VH) domain as provided herein including embodimentsthereof may each independently form part of an antibody, an antibodyvariant, a fragment of an antibody, a fragment of an antibody variant,or a recombinant protein (e.g., a chimeric antigen receptor, bispecificantibody). Provided herein are recombinant proteins (e.g., a chimericantigen receptor, a bispecific antibody) which include the light chainvariable (VL) domain and the heavy chain variable (VH) domain asprovided herein and are therefore capable of binding IL1RAP andrecruiting effector cells to IL1RAP-expressing cells (e.g., LSCs)thereby eliminating the IL1RAP-expressing cells. In embodiments, therecombinant protein is a chimeric antigen receptor (CAR). Inembodiments, the recombinant protein is a bispecific antibody.

The light chain variable (VL) domain and the heavy chain variable (VH)domain as provided herein may form part of a chimeric antigen receptor.Thus, in an aspect is provided a recombinant protein including: (i) anantibody region including: (a) a light chain variable domain including aCDR L1 as set forth in SEQ ID NO:1, a CDR L2 as set forth in SEQ ID NO:2and a CDR L3 as set forth in SEQ ID NO:3; and (b) a heavy chain variableregion domain a CDR H1 as set forth in SEQ ID NO:4, a CDR H2 as setforth in SEQ ID NO:5, and a CDR H3 as set forth in SEQ ID NO:6; and (ii)a transmembrane domain.

An “antibody region” as provided herein refers to a monovalent ormultivalent protein moiety that forms part of the recombinant protein(e.g., CAR) provided herein including embodiments thereof. A person ofordinary skill in the art would therefore immediately recognize that theantibody region is a protein moiety capable of binding an antigen(epitope). Thus, the antibody region provided herein may include adomain of an antibody (e.g., a light chain variable (VL) domain, a heavychain variable (VH) domain) or a fragment of an antibody (e.g., Fab). Inembodiments, the antibody region is a protein conjugate. A “proteinconjugate” as provided herein refers to a construct consisting of morethan one polypeptide, wherein the polypeptides are bound togethercovalently or non-covalently. In embodiments, the protein conjugateincludes a Fab moiety (a monovalent Fab) covalently attached to an scFvmoiety (a monovalent scFv). In embodiments, the protein conjugateincludes a plurality (at least two) Fab moieties. In embodiments, thepolypeptides of a protein conjugate are encoded by one nucleic acidmolecule. In embodiments, the polypeptides of a protein conjugate areencoded by different nucleic acid molecules. In embodiments, thepolypeptides are connected through a linker. In embodiments, thepolypeptides are connected through a chemical linker. In embodiments,the antibody region is an scFv.

A “transmembrane domain” as provided herein refers to a polypeptideforming part of a biological membrane. The transmembrane domain providedherein is capable of spanning a biological membrane (e.g., a cellularmembrane) from one side of the membrane through to the other side of themembrane. In embodiments, the transmembrane domain spans from theintracellular side to the extracellular side of a cellular membrane.Transmembrane domains may include non-polar, hydrophobic residues, whichanchor the proteins provided herein including embodiments thereof in abiological membrane (e.g., cellular membrane of a T cell). Anytransmembrane domain capable of anchoring the proteins provided hereinincluding embodiments thereof are contemplated. Non-limiting examples oftransmembrane domains include, the transmembrane domains of CD28, CD8,CD4 or CD3-zeta. In embodiments, the transmembrane domain is a CD4transmembrane domain.

In embodiments, the transmembrane domain is a CD28 transmembrane domain.The term “CD28 transmembrane domain” as provided herein includes any ofthe recombinant or naturally-occurring forms of the transmembrane domainof CD28, or variants or homologs thereof that maintain CD28transmembrane domain activity (e.g. within at least 50%, 80%, 90%, 95%,96%, 97%, 98%, 99% or 100% activity compared to the CD28 transmembranedomain). In some aspects, the variants or homologs have at least 90%,95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across thewhole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200continuous amino acid portion) compared to a naturally occurring CD28transmembrane domain polypeptide. In embodiments, CD28 is the protein asidentified by the NCBI sequence reference GI:340545506, homolog orfunctional fragment thereof.

In embodiments, the transmembrane domain is a CD8 transmembrane domain.The term “CD8 transmembrane domain” as provided herein includes any ofthe recombinant or naturally-occurring forms of the transmembrane domainof CD8, or variants or homologs thereof that maintain CD8 transmembranedomain activity (e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%,99% or 100% activity compared to the CD8 transmembrane domain). In someaspects, the variants or homologs have at least 90%, 95%, 96%, 97%, 98%,99% or 100% amino acid sequence identity across the whole sequence or aportion of the sequence (e.g. a 50, 100, 150 or 200 continuous aminoacid portion) compared to a naturally occurring CD8 transmembrane domainpolypeptide. In embodiments, CD8 is the protein as identified by theNCBI sequence reference GI:225007534, homolog or functional fragmentthereof.

In embodiments, the transmembrane domain is a CD4 transmembrane domain.The term “CD4 transmembrane domain” as provided herein includes any ofthe recombinant or naturally-occurring forms of the transmembrane domainof CD4, or variants or homologs thereof that maintain CD4 transmembranedomain activity (e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%,99% or 100% activity compared to the CD4 transmembrane domain). In someaspects, the variants or homologs have at least 90%, 95%, 96%, 97%, 98%,99% or 100% amino acid sequence identity across the whole sequence or aportion of the sequence (e.g. a 50, 100, 150 or 200 continuous aminoacid portion) compared to a naturally occurring CD4 transmembrane domainpolypeptide. In embodiments, CD4 is the protein as identified by theNCBI sequence reference GI:303522473, homolog or functional fragmentthereof.

In embodiments, the transmembrane domain is a CD3-zeta (also known asCD247) transmembrane domain. The term “CD3-zeta transmembrane domain” asprovided herein includes any of the recombinant or naturally-occurringforms of the transmembrane domain of CD3-zeta, or variants or homologsthereof that maintain CD3-zeta transmembrane domain activity (e.g.within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activitycompared to the CD3-zeta transmembrane domain). In some aspects, thevariants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100%amino acid sequence identity across the whole sequence or a portion ofthe sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion)compared to a naturally occurring CD3-zeta transmembrane domainpolypeptide. In embodiments, CD3-zeta is the protein as identified bythe NCBI sequence reference GI:166362721, homolog or functional fragmentthereof.

In embodiments, light chain variable domain includes the sequence of SEQID NO:7. In embodiments, light chain variable domain is the sequence ofSEQ ID NO:7. In embodiments, the heavy chain variable domain incudes thesequence of SEQ ID NO:8. In embodiments, the heavy chain variable domainis the sequence of SEQ ID NO:8. In embodiments, the light chain variabledomain includes a FR L1 as set forth in SEQ ID NO:9, a FR L2 as setforth in SEQ ID NO:10, FR L3 as set forth in SEQ ID NO:11 and a FR L4 asset forth in SEQ ID NO:12. In embodiments, the heavy chain variabledomain includes a FR H1 as set forth in SEQ ID NO:13, a FR H2 as setforth in SEQ ID NO:14, FR H3 as set forth in SEQ ID NO:15 and a FR H4 asset forth in SEQ ID NO:16.

In embodiments, the antibody region includes a single-chain variablefragment (scFv). In embodiments, the scFv includes the sequence of SEQID NO:17. In embodiments, the scFv is the sequence of SEQ ID NO:17.

In embodiments, the recombinant protein is capable of binding IL1RAPwith an equilibrium dissociation constant (K_(D)) of about 21 nM. Inembodiments, the recombinant protein is capable of binding IL1RAP withan equilibrium dissociation constant (K_(D)) of 21 nM. The K_(D) valuesdefined herein for the IL1RAP antibody do apply for the recombinantproteins described herein including embodiments thereof.

Thus, in embodiments, the recombinant protein (e.g., chimeric antigenreceptor) has an EC₅₀ (half maximal effective concentration) from about1 ng/ml to about 20 ng/ml. The term “EC50” or “half maximal effectiveconcentration” as used herein refers to the concentration of a molecule(e.g., antibody, chimeric antigen receptor or bispecific antibody)capable of inducing a response which is halfway between the baselineresponse and the maximum response after a specified exposure time. Inembodiments, the EC50 is the concentration of a molecule (e.g.,antibody, chimeric antigen receptor or bispecific antibody) thatproduces 50% of the maximal possible effect of that molecule. Inembodiments, the recombinant protein has an EC₅₀ from about 2 ng/ml toabout 20 ng/ml. In embodiments, the recombinant protein has an EC₅₀ fromabout 3 ng/ml to about 20 ng/ml. In embodiments, the recombinant proteinhas an EC₅₀ from about 4 ng/ml to about 20 ng/ml. In embodiments, therecombinant protein has an EC₅₀ from about 5 ng/ml to about 20 ng/ml. Inembodiments, the recombinant protein has an EC₅₀ from about 6 ng/ml toabout 20 ng/ml. In embodiments, the recombinant protein has an EC₅₀ fromabout 7 ng/ml to about 20 ng/ml. In embodiments, the recombinant proteinhas an EC₅₀ from about 8 ng/ml to about 20 ng/ml. In embodiments, therecombinant protein has an EC₅₀ from about 9 ng/ml to about 20 ng/ml. Inembodiments, the recombinant protein has an EC₅₀ from about 10 ng/ml toabout 20 ng/ml.

In embodiments, the recombinant protein has an EC₅₀ from about 11 ng/mlto about 20 ng/ml. In embodiments, the recombinant protein has an EC₅₀from about 12 ng/ml to about 20 ng/ml. In embodiments, the recombinantprotein has an EC₅₀ from about 13 ng/ml to about 20 ng/ml. Inembodiments, the recombinant protein has an EC₅₀ from about 14 ng/ml toabout 20 ng/ml. In embodiments, the recombinant protein has an EC₅₀ fromabout 15 ng/ml to about 20 ng/ml. In embodiments, the recombinantprotein has an EC₅₀ from about 16 ng/ml to about 20 ng/ml. Inembodiments, the recombinant protein has an EC₅₀ from about 17 ng/ml toabout 20 ng/ml. In embodiments, the recombinant protein has an EC₅₀ fromabout 18 ng/ml to about 20 ng/ml. In embodiments, the recombinantprotein has an EC₅₀ from about 19 ng/ml to about 20 ng/ml.

In embodiments, the recombinant protein has an EC₅₀ of about 1 ng/ml, 2ng/ml, 4 ng/ml, 5 ng/ml, 6 ng/ml, 7 ng/ml, 8 ng/ml, 9 ng/ml, 10 ng/ml,11 ng/ml, 12 ng/ml, 13 ng/ml, 14 ng/ml, 15 ng/ml, 16 ng/ml, 17 ng/ml, 18ng/ml, 19 ng/ml or 20 ng/ml. In embodiments, the antibody has an EC₅₀ of1 ng/ml, 2 ng/ml, 4 ng/ml, 5 ng/ml, 6 ng/ml, 7 ng/ml, 8 ng/ml, 9 ng/ml,10 ng/ml, 11 ng/ml, 12 ng/ml, 13 ng/ml, 14 ng/ml, 15 ng/ml, 16 ng/ml, 17ng/ml, 18 ng/ml, 19 ng/ml or 20 ng/ml.

In embodiments, the recombinant protein has an EC₅₀ from about 1 toabout 19 ng/ml. In embodiments, the recombinant protein has an EC₅₀ fromabout 1 ng/ml to about 18 ng/ml. In embodiments, the recombinant proteinhas an EC₅₀ from about 1 ng/ml to about 17 ng/ml. In embodiments, therecombinant protein has an EC₅₀ from about 1 ng/ml to about 16 ng/ml. Inembodiments, the recombinant protein has an EC₅₀ from about 1 ng/ml toabout 15 ng/ml. In embodiments, the recombinant protein has an EC₅₀ fromabout 1 ng/ml to about 14 ng/ml. In embodiments, the recombinant proteinhas an EC₅₀ from about 1 ng/ml to about 13 ng/ml. In embodiments, therecombinant protein has an EC₅₀ from about 1 ng/ml to about 12 ng/ml. Inembodiments, the recombinant protein has an EC₅₀ from about 1 ng/ml toabout 11 ng/ml.

In embodiments, the recombinant protein has an EC₅₀ from about 1 ng/mlto about 10 ng/ml. In embodiments, the recombinant protein has an EC₅₀from about 1 ng/ml to about 9 ng/ml. In embodiments, the recombinantprotein has an EC₅₀ from about 1 ng/ml to about 8 ng/ml. In embodiments,the recombinant protein has an EC₅₀ from about 1 ng/ml to about 7 ng/ml.In embodiments, the recombinant protein has an EC₅₀ from about 1 ng/mlto about 6 ng/ml. In embodiments, the recombinant protein has an EC₅₀from about 1 ng/ml to about 5 ng/ml. In embodiments, the recombinantprotein has an EC₅₀ from about 1 ng/ml to about 4 ng/ml. In embodiments,the recombinant protein has an EC₅₀ from about 1 ng/ml to about 3 ng/ml.In embodiments, the recombinant protein has an EC₅₀ from about 1 ng/mlto about 2 ng/ml.

In embodiments, the recombinant protein has an EC₅₀ from about 4 ng/mlto about 7 ng/ml. In embodiments, the recombinant protein has an EC₅₀from about 4.5 ng/ml to about 7 ng/ml. In embodiments, the recombinantprotein has an EC₅₀ from about 5 ng/ml to about 7 ng/ml. In embodiments,the recombinant protein has an EC₅₀ from about 5.5 ng/ml to about 7ng/ml. In embodiments, the recombinant protein has an EC₅₀ from about 6ng/ml to about 7 ng/ml. In embodiments, the recombinant protein has anEC₅₀ from about 6.5 ng/ml to about 7 ng/ml.

In embodiments, the recombinant protein has an EC₅₀ from 4 ng/ml to 7ng/ml. In embodiments, the recombinant protein has an EC₅₀ from 4.5ng/ml to 7 ng/ml. In embodiments, the recombinant protein has an EC₅₀from 5 ng/ml to 7 ng/ml. In embodiments, the recombinant protein has anEC₅₀ from 5.5 ng/ml to 7 ng/ml. In embodiments, the recombinant proteinhas an EC₅₀ from 6 ng/ml to 7 ng/ml. In embodiments, the recombinantprotein has an EC₅₀ from 6.5 ng/ml to 7 ng/ml.

In embodiments, the recombinant protein has an EC₅₀ of about 4.9 ng/ml.In embodiments, the recombinant protein has an EC₅₀ of 4.9 ng/ml. Inembodiments, the recombinant protein has an EC₅₀ of about 6.8 ng/ml. Inembodiments, the recombinant protein has an EC₅₀ of 6.8 ng/ml. Inembodiments, the recombinant protein has an EC₅₀ of about 6.4 ng/ml. Inembodiments, the recombinant protein has an EC₅₀ of 6.4 ng/ml. Inembodiments, the recombinant protein has an EC₅₀ of about 5.4 ng/ml. Inembodiments, the recombinant protein has an EC₅₀ of 5.4 ng/ml.

In embodiments, the recombinant protein is bound to an IL1RAP. Inembodiments, the IL1RAP is a human IL1RAP. In embodiments, the IL1RAPforms part of a cell. In embodiments, the IL1RAP is expressed on thesurface of the cell. In embodiments, the cell is a cancer cell. Inembodiments, the cancer cell is a leukemia stem cell (LSC). Inembodiments, the cancer cell is an acute myeloid leukemia (AML) cell. Inembodiments, the cancer cell is a chronic myeloid leukemia (CML) cell.In embodiments, the cancer cell is a lung cancer cell. In embodiments,the cancer cell is a non-small cell lung cancer (NSCLC) cell. Inembodiments, the cancer cell is a pancreatic cancer cell. Inembodiments, the cancer cell is a melanoma cell. In embodiments, thecancer cell is a breast cancer cell. In embodiments, the cancer cell isa colon cancer cell.

In embodiments, the antibody region includes an Fc domain. Inembodiments, the Fc domain is an IgG4 Fc domain. In embodiments, theantibody region includes an Fc hinge domain. In embodiments, theantibody region includes an IgG4 Fc hinge domain. In embodiments, theantibody region includes a spacer region. In embodiments, the spacerregion is between the transmembrane domain and the antibody region. A“spacer region” as provided herein is a polypeptide connecting theantibody region with the transmembrane domain. In embodiments, thespacer region connects the heavy chain constant region with thetransmembrane domain. In embodiments, the spacer region includes an Fcregion. In embodiments, the spacer region is an Fc region. Examples ofspacer regions contemplated for the recombinant protein compositionsprovided herein include without limitation, immunoglobulin molecules orfragments thereof (e.g., IgG1, IgG2, IgG3, IgG4) and immunoglobulinmolecules or fragments thereof (e.g., IgG1, IgG2, IgG3, IgG4) includingmutations affecting Fc receptor binding. In embodiments, the spacerregion is a hinge region. In embodiments, the spacer region is an IgG4hinge region. In embodiments, the spacer region is a modified IgG4 hingeregion.

In embodiments, the recombinant protein as provided herein, includingembodiments thereof, further includes an intracellular co-stimulatorysignaling domain. An “intracellular co-stimulatory signaling domain” asprovided herein includes amino acid sequences capable of providingco-stimulatory signaling in response to binding of an antigen to theantibody region provided herein including embodiments thereof. Inembodiments, the signaling of the co-stimulatory signaling domainresults in production of cytokines and proliferation of the T cellexpressing the same. In embodiments, the intracellular co-stimulatorysignaling domain is a CD28 intracellular co-stimulatory signalingdomain, a 4-1BB intracellular co-stimulatory signaling domain, a ICOSintracellular co-stimulatory signaling domain, or an OX-40 intracellularco-stimulatory signaling domain. In embodiments, the intracellularco-stimulatory signaling domain is a CD28 intracellular co-stimulatorysignaling domain. In embodiments, the intracellular co-stimulatorysignaling domain is a 4-1BB intracellular co-stimulatory signalingdomain. In embodiments, the intracellular co-stimulatory signalingdomain is a ICOS intracellular co-stimulatory signaling domain. Inembodiments, the intracellular co-stimulatory signaling domain is anOX-40 intracellular co-stimulatory signaling domain.

In embodiments, the recombinant protein as provided herein includingembodiments thereof, further includes an intracellular T-cell signalingdomain. An “intracellular T-cell signaling domain” as provided hereinincludes amino acid sequences capable of providing primary signaling inresponse to binding of an antigen to the antibody region provided hereinincluding embodiments thereof. In embodiments, the signaling of theintracellular T-cell signaling domain results in activation of the Tcell expressing the same. In embodiments, the signaling of theintracellular T-cell signaling domain results in proliferation (celldivision) of the T cell expressing the same. In embodiments, thesignaling of the intracellular T-cell signaling domain resultsexpression by said T cell of proteins known in the art to characteristicof activated T cell (e.g., CTLA-4, PD-1, CD28, CD69). In embodiments,the intracellular T-cell signaling domain includes the signaling domainof the zeta chain of the human CD3 complex. In embodiments, theintracellular T-cell signaling domain is a CD3 (intracellular T-cellsignaling domain.

The term “CTLA-4” as referred to herein includes any of the recombinantor naturally-occurring forms of the cytotoxic T-lymphocyte-associatedprotein 4 protein, also known as CD152 (cluster of differentiation 152),or variants or homologs thereof that maintain CTLA-4 activity (e.g.within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activitycompared to CTLA-4). In some aspects, the variants or homologs have atleast 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identityacross the whole sequence or a portion of the sequence (e.g. a 50, 100,150 or 200 continuous amino acid portion) compared to a naturallyoccurring CTLA-4 protein. In embodiments, the CTLA-4 protein issubstantially identical to the protein identified by the UniProtreference number P16410 or a variant or homolog having substantialidentity thereto.

The term “PD-1” as referred to herein includes any of the recombinant ornaturally-occurring forms of the Programmed cell death protein 1protein, also known as CD279 (cluster of differentiation 279), orvariants or homologs thereof that maintain PD-1 activity (e.g. within atleast 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity comparedto PD-1). In some aspects, the variants or homologs have at least 90%,95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across thewhole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200continuous amino acid portion) compared to a naturally occurring PD-1protein. In embodiments, the PD-1 protein is substantially identical tothe protein identified by the UniProt reference number Q15116 or avariant or homolog having substantial identity thereto.

The term “CD28” as referred to herein includes any of the recombinant ornaturally-occurring forms of the Cluster of Differentiation 28 protein,or variants or homologs thereof that maintain CD28 activity (e.g. withinat least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activitycompared to CD28). In some aspects, the variants or homologs have atleast 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identityacross the whole sequence or a portion of the sequence (e.g. a 50, 100,150 or 200 continuous amino acid portion) compared to a naturallyoccurring CD28 protein. In embodiments, the CD28 protein issubstantially identical to the protein identified by the UniProtreference number P10747 or a variant or homolog having substantialidentity thereto.

The term “CD69” as referred to herein includes any of the recombinant ornaturally-occurring forms of the Cluster of Differentiation 69 protein,or variants or homologs thereof that maintain CD69 activity (e.g. withinat least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activitycompared to CD69). In some aspects, the variants or homologs have atleast 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identityacross the whole sequence or a portion of the sequence (e.g. a 50, 100,150 or 200 continuous amino acid portion) compared to a naturallyoccurring CD69 protein. In embodiments, the CD69 protein issubstantially identical to the protein identified by the UniProtreference number Q07108 or a variant or homolog having substantialidentity thereto.

The term “4-1BB” as referred to herein includes any of the recombinantor naturally-occurring forms of the 4-1BB protein, also known as tumornecrosis factor receptor superfamily member 9 (TNFRSF9), Cluster ofDifferentiation 137 (CD137) and induced by lymphocyte activation (ILA),or variants or homologs thereof that maintain 4-1BB activity (e.g.within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activitycompared to 4-1BB). In some aspects, the variants or homologs have atleast 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identityacross the whole sequence or a portion of the sequence (e.g. a 50, 100,150 or 200 continuous amino acid portion) compared to a naturallyoccurring EGFR protein. In embodiments, the 4-1BB protein issubstantially identical to the protein identified by the UniProtreference number Q07011 or a variant or homolog having substantialidentity thereto.

In embodiments, the recombinant protein as provided herein includingembodiments thereof, further includes a self-cleaving peptidyl sequence.In embodiments, the self-cleaving peptidyl linker sequence is a T2Asequence or a 2A sequence. In embodiments, the self-cleaving peptidyllinker sequence is a T2A sequence. In embodiments, the self-cleavingpeptidyl linker sequence is a 2A sequence.

In embodiments, the recombinant protein as provided herein includingembodiments thereof, further includes a detectable domain. A “detectabledomain” as provided herein is peptide moiety detectable byspectroscopic, photochemical, biochemical, immunochemical, chemical, orother physical means. For example, a detectable domain as providedherein may be a protein or other entity which can be made detectable,e.g., by incorporating a radiolabel or being reactive to an antibodyspecifically. Any appropriate method known in the art for conjugating anantibody to the label may be employed, e.g., using methods described inHermanson, Bioconjugate Techniques 1996, Academic Press, Inc., SanDiego. In embodiments, the detectable domain is a truncated EGFR (EGFRt)domain. The term “EGFRt” refers to a truncated epidermal growth factorreceptor protein lacking intracellular signaling capabilities. As usedherein, EGFRt is an inert cell surface molecule which functions as adetectable domain allowing identification of transduced T cells. Inembodiments, the recombinant protein forms part of a cell. Inembodiments, the recombinant protein forms part of a T cell.

The term “EGFR” as referred to herein includes any of the recombinant ornaturally-occurring forms of the epidermal growth factor receptorprotein, also known as ErbB-1 and HER1, or variants or homologs thereofthat maintain EGFR activity (e.g. within at least 50%, 80%, 90%, 95%,96%, 97%, 98%, 99% or 100% activity compared to EGFR). In some aspects,the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or100% amino acid sequence identity across the whole sequence or a portionof the sequence (e.g. a 50, 100, 150 or 200 continuous amino acidportion) compared to a naturally occurring EGFR protein. In embodiments,the EGFR protein is substantially identical to the protein identified bythe UniProt reference number P00533 or a variant or homolog havingsubstantial identity thereto.

In one embodiment, the recombinant protein includes a light chainvariable domain of SEQ ID NO:7, a heavy chain variable domain of SEQ IDNO:8, wherein the transmembrane domain is a CD4 transmembrane domain,the Fc domain is an IgG4 hinge domain, the intracellular co-stimulatorydomain is a 4-1BB intracellular co-stimulatory domain, the intracellularT-cell signaling domain is a CD3ζ intracellular T-cell signaling domain,the self-cleaving peptidyl sequence is a T2A sequence, and thedetectable domain is a truncated EGFR domain.

In embodiments, the recombinant protein is a chimeric antigen receptor,including an antibody region of SEQ ID NO:17, a CD4 transmembranedomain, a IgG4 hinge domain, a 4-1BB intracellular co-stimulatorydomain, a CD3ζ intracellular T-cell signaling domain, a T2Aself-cleaving peptidyl sequence and a truncated EGFR domain.

In embodiments, the recombinant protein is a chimeric antigen receptor,including an antibody region of SEQ ID NO:17, a CD8 hinge and a CD8transmembrane domains, a 4-1BB intracellular co-stimulatory domain, anda CD3ζ intracellular T-cell signaling domain.

In embodiments, the recombinant protein is a chimeric antigen receptorof SEQ ID NO:90.

The light chain variable (VL) domain and the heavy chain variable (VH)domain as provided herein may form part of a bispecific antibody. Thus,in another aspect is provided a recombinant protein including: (i) afirst antibody region capable of binding an effector cell ligand; and(ii) a second antibody region, including: (a) a light chain variabledomain comprising a CDR L1 as set forth in SEQ ID NO:1, a CDR L2 as setforth in SEQ ID NO:2 and a CDR L3 as set forth in SEQ ID NO:3; and (b) aheavy chain variable region domain a CDR H1 as set forth in SEQ ID NO:4,a CDR H2 as set forth in SEQ ID NO:5, and a CDR H3 as set forth in SEQID NO:6.

The term “effector cell ligand” as provided herein refers to a cellsurface molecule expressed on an effector cell of the immune system(e.g., a cytotoxic T cell, a helper T cell, a B cell, a natural killercell). Upon binding of the first antibody region to the effector cellligand expressed on the effector cell, the effector cell is activatedand able to exert its function (e.g., selective killing or eradicationof malignant, infected or otherwise unhealthy cells). In embodiments,the effector cell ligand is a CD3 protein. In embodiments, the effectorcell ligand is a CD16 protein. In embodiments, the effector cell ligandis a CD32 protein. In embodiments, the effector cell ligand is a NKp46protein. The first antibody region as provided herein may be anantibody, an antibody variant, a fragment of an antibody or a fragmentof an antibody variant.

A “CD3 protein” as referred to herein includes any of the recombinant ornaturally-occurring forms of the Cluster of Differentiation 3 (CD3)proteins or variants or homologs thereof that comprise the CD3 complexthat mediates signal transduction and maintains CD3 complex activity(e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100%activity compared to the CD3 complex). In some aspects, the variants orhomologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acidsequence identity across the whole sequence or a portion of the sequence(e.g. a 50, 100, 150 or 200 continuous amino acid portion) compared to anaturally occurring CD3 proteins in the CD3 complex.

A “CD16 protein” as referred to herein includes any of the recombinantor naturally-occurring forms of the Cluster of Differentiation 16 (CD16)protein, also known as low affinity immunoglobulin gamma Fc regionreceptor III-A, or variants or homologs thereof that maintain CD16activity (e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or100% activity compared to CD16). In some aspects, the variants orhomologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acidsequence identity across the whole sequence or a portion of the sequence(e.g. a 50, 100, 150 or 200 continuous amino acid portion) compared to anaturally occurring CD16 protein. In embodiments, the CD16 protein issubstantially identical to the protein identified by the UniProtreference number P08637 or a variant or homolog having substantialidentity thereto.

A “CD32 protein” as referred to herein includes any of the recombinantor naturally-occurring forms of the Cluster of Differentiation 32 (CD32)protein, also known as low affinity immunoglobulin gamma Fc regionreceptor II-A, or variants or homologs thereof that maintain CD32activity (e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or100% activity compared to CD32). In some aspects, the variants orhomologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acidsequence identity across the whole sequence or a portion of the sequence(e.g. a 50, 100, 150 or 200 continuous amino acid portion) compared to anaturally occurring CD32 protein. In embodiments, the CD32 protein issubstantially identical to the protein identified by the UniProtreference number P12318 or a variant or homolog having substantialidentity thereto.

A “NKp46 protein” as referred to herein includes any of the recombinantor naturally-occurring forms of the NKp46 protein, also known as naturalcytotoxicity triggering receptor 1, or variants or homologs thereof thatmaintain NKp46 activity (e.g. within at least 50%, 80%, 90%, 95%, 96%,97%, 98%, 99% or 100% activity compared to NKp46). In some aspects, thevariants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100%amino acid sequence identity across the whole sequence or a portion ofthe sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion)compared to a naturally occurring NKp46 protein. In embodiments, theNKp46 protein is substantially identical to the protein identified bythe UniProt reference number 076036 or a variant or homolog havingsubstantial identity thereto.

In embodiments, the recombinant protein (e.g., bispecific antibody) hasan IC50 (half maximal inhibitory concentration) from about 10 pM toabout 100 pM. In embodiments, the recombinant protein has an IC50 fromabout 15 pM to about 100 pM. In embodiments, the recombinant protein hasan IC50 from about 20 pM to about 100 pM. In embodiments, therecombinant protein has an IC50 from about 25 pM to about 100 pM. Inembodiments, the recombinant protein has an IC50 from about 30 pM toabout 100 pM. In embodiments, the recombinant protein has an IC50 fromabout 35 pM to about 100 pM. In embodiments, the recombinant protein hasan IC50 from about 40 pM to about 100 pM. In embodiments, therecombinant protein has an IC50 from about 45 pM to about 100 pM. Inembodiments, the recombinant protein has an IC50 from about 50 pM toabout 100 pM. In embodiments, the recombinant protein has an IC50 fromabout 55 pM to about 100 pM. In embodiments, the recombinant protein hasan IC50 from about 60 pM to about 100 pM. In embodiments, therecombinant protein has an IC50 from about 65 pM to about 100 pM. Inembodiments, the recombinant protein has an IC50 from about 70 pM toabout 100 pM. In embodiments, the recombinant protein has an IC50 fromabout 75 pM to about 100 pM. In embodiments, the recombinant protein hasan IC50 from about 80 pM to about 100 pM. In embodiments, therecombinant protein has an IC50 from about 85 pM to about 100 pM. Inembodiments, the recombinant protein has an IC50 from about 90 pM toabout 100 pM. In embodiments, the recombinant protein has an IC50 fromabout 95 pM to about 100 pM.

In embodiments, the recombinant protein has an IC50 (the half maximalinhibitory concentration) from 10 pM to 100 pM. In embodiments, therecombinant protein has an IC50 from 15 pM to 100 pM. In embodiments,the recombinant protein has an IC50 from 20 pM to 100 pM. Inembodiments, the recombinant protein has an IC50 from 25 pM to 100 pM.In embodiments, the recombinant protein has an IC50 from 30 pM to 100pM. In embodiments, the recombinant protein has an IC50 from 35 pM to100 pM. In embodiments, the recombinant protein has an IC50 from 40 pMto 100 pM. In embodiments, the recombinant protein has an IC50 from 45pM to 100 pM. In embodiments, the recombinant protein has an IC50 from50 pM to 100 pM. In embodiments, the recombinant protein has an IC50from 55 pM to 100 pM. In embodiments, the recombinant protein has anIC50 from 60 pM to 100 pM. In embodiments, the recombinant protein hasan IC50 from 65 pM to 100 pM. In embodiments, the recombinant proteinhas an IC50 from 70 pM to 100 pM. In embodiments, the recombinantprotein has an IC50 from 75 pM to 100 pM. In embodiments, therecombinant protein has an IC50 from 80 pM to 100 pM. In embodiments,the recombinant protein has an IC50 from 85 pM to 100 pM. Inembodiments, the recombinant protein has an IC50 from 90 pM to 100 pM.In embodiments, the recombinant protein has an IC50 from 95 pM to 100pM.

In embodiments, the recombinant protein has an IC50 from about 40 toabout 50 pM. In embodiments, the recombinant protein has an IC50 fromabout 41 to about 50 pM. In embodiments, the recombinant protein has anIC50 from about 42 to about 50 pM. In embodiments, the recombinantprotein has an IC50 from about 43 to about 50 pM. In embodiments, therecombinant protein has an IC50 from about 44 to about 50 pM. Inembodiments, the recombinant protein has an IC50 from about 45 to about50 pM. In embodiments, the recombinant protein has an IC50 from about 46to about 50 pM. In embodiments, the recombinant protein has an IC50 fromabout 47 to about 50 pM. In embodiments, the recombinant protein has anIC50 from about 48 to about 50 pM. In embodiments, the recombinantprotein has an IC50 from about 49 to about 50 pM.

In embodiments, the recombinant protein has an IC50 from about 40 toabout 49 pM. In embodiments, the recombinant protein has an IC50 fromabout 40 to about 48 pM. In embodiments, the recombinant protein has anIC50 from about 40 to about 47 pM. In embodiments, the recombinantprotein has an IC50 from about 40 to about 46 pM. In embodiments, therecombinant protein has an IC50 from about 40 to about 45 pM. Inembodiments, the recombinant protein has an IC50 from about 40 to about44 pM. In embodiments, the recombinant protein has an IC50 from about 40to about 43 pM. In embodiments, the recombinant protein has an IC50 fromabout 40 to about 42 pM. In embodiments, the recombinant protein has anIC50 from about 40 to about 41 pM.

In embodiments, the recombinant protein has an IC50 from 40 to 50 pM. Inembodiments, the recombinant protein has an IC50 from 41 to 50 pM. Inembodiments, the recombinant protein has an IC50 from 42 to 50 pM. Inembodiments, the recombinant protein has an IC50 from 43 to 50 pM. Inembodiments, the recombinant protein has an IC50 from 44 to 50 pM. Inembodiments, the recombinant protein has an IC50 from 45 to 50 pM. Inembodiments, the recombinant protein has an IC50 from 46 to 50 pM. Inembodiments, the recombinant protein has an IC50 from 47 to 50 pM. Inembodiments, the recombinant protein has an IC50 from 48 to 50 pM. Inembodiments, the recombinant protein has an IC50 from 49 to 50 pM.

In embodiments, the recombinant protein has an IC50 from 40 to 49 pM. Inembodiments, the recombinant protein has an IC50 from 40 to 48 pM. Inembodiments, the recombinant protein has an IC50 from 40 to 47 pM. Inembodiments, the recombinant protein has an IC50 from 40 to 46 pM. Inembodiments, the recombinant protein has an IC50 from 40 to 45 pM. Inembodiments, the recombinant protein has an IC50 from 40 to 44 pM. Inembodiments, the recombinant protein has an IC50 from 40 to 43 pM. Inembodiments, the recombinant protein has an IC50 from 40 to 42 pM. Inembodiments, the recombinant protein has an IC50 from 40 to 41 pM.

In embodiments, the recombinant protein has an IC50 from 40 to 48.5 pM.In embodiments, the recombinant protein has an IC50 from 40 to 47.5 pM.In embodiments, the recombinant protein has an IC50 from 40 to 46.5 pM.In embodiments, the recombinant protein has an IC50 from 40 to 45.5 pM.In embodiments, the recombinant protein has an IC50 from 40 to 44.5 pM.In embodiments, the recombinant protein has an IC50 from 40 to 43.5 pM.In embodiments, the recombinant protein has an IC50 from 40 to 42.5 pM.In embodiments, the recombinant protein has an IC50 from 40 to 41.5 pM.In embodiments, the recombinant protein has an IC50 from 40 to 40.5 pM.

In embodiments, the recombinant protein has an IC50 from 40.5 to 49 pM.In embodiments, the recombinant protein has an IC50 from 40.5 to 48 pM.In embodiments, the recombinant protein has an IC50 from 40.5 to 47 pM.In embodiments, the recombinant protein has an IC50 from 40.5 to 46 pM.In embodiments, the recombinant protein has an IC50 from 40.5 to 45 pM.In embodiments, the recombinant protein has an IC50 from 40.5 to 44 pM.In embodiments, the recombinant protein has an IC50 from 40.5 to 43 pM.In embodiments, the recombinant protein has an IC50 from 40.5 to 42 pM.In embodiments, the recombinant protein has an IC50 from 40.5 to 41 pM.

In embodiments, the recombinant protein has an IC50 from 41.5 to 49 pM.In embodiments, the recombinant protein has an IC50 from 41.5 to 48 pM.In embodiments, the recombinant protein has an IC50 from 41.5 to 47 pM.In embodiments, the recombinant protein has an IC50 from 41.5 to 46 pM.In embodiments, the recombinant protein has an IC50 from 41.5 to 45 pM.In embodiments, the recombinant protein has an IC50 from 41.5 to 44 pM.In embodiments, the recombinant protein has an IC50 from 41.5 to 43 pM.In embodiments, the recombinant protein has an IC50 from 41.5 to 42 pM.

In embodiments, the recombinant protein has an IC50 from 42.5 to 49 pM.In embodiments, the recombinant protein has an IC50 from 42.5 to 48 pM.In embodiments, the recombinant protein has an IC50 from 42.5 to 47 pM.In embodiments, the recombinant protein has an IC50 from 42.5 to 46 pM.In embodiments, the recombinant protein has an IC50 from 42.5 to 45 pM.In embodiments, the recombinant protein has an IC50 from 42.5 to 44 pM.In embodiments, the recombinant protein has an IC50 from 42.5 to 43 pM.

In embodiments, the recombinant protein has an IC50 from 43.5 to 49 pM.In embodiments, the recombinant protein has an IC50 from 43.5 to 48 pM.In embodiments, the recombinant protein has an IC50 from 43.5 to 47 pM.In embodiments, the recombinant protein has an IC50 from 43.5 to 46 pM.In embodiments, the recombinant protein has an IC50 from 43.5 to 45 pM.In embodiments, the recombinant protein has an IC50 from 43.5 to 44 pM.

In embodiments, the recombinant protein has an IC50 from 44.5 to 49 pM.In embodiments, the recombinant protein has an IC50 from 44.5 to 48 pM.In embodiments, the recombinant protein has an IC50 from 44.5 to 47 pM.In embodiments, the recombinant protein has an IC50 from 44.5 to 46 pM.In embodiments, the recombinant protein has an IC50 from 44.5 to 45 pM.

In embodiments, the recombinant protein has an IC50 of about 10 pM, 20pM, 30 pM, 40 pM, 41 pM, 42 pM, 43 pM, 44 pM, 45 pM, 46 pM, 47 pM, 48pM, 49 pM, 50 pM, 60 pM, 70 pM, 80 pM, 90 pM, or 100 pM. In embodiments,the recombinant protein has an IC50 of 10 pM, 20 pM, 30 pM, 40 pM, 41pM, 42 pM, 43 pM, 44 pM, 45 pM, 46 pM, 47 pM, 48 pM, 49 pM, 50 pM, 60pM, 70 pM, 80 pM, 90 pM, or 100 pM.

In embodiments, the recombinant protein has an IC50 of about 45.14 pM.In embodiments, the recombinant protein has an IC50 of 45.14 pM. Inembodiments, the recombinant protein has an IC50 of about 45.14 pM. Inembodiments, the recombinant protein has an IC50 of 45.14 pM.

In embodiments, the light chain variable domain includes the sequence ofSEQ ID NO:7. In embodiments, the light chain variable domain is thesequence of SEQ ID NO:7. In embodiments, the heavy chain variable domainincludes the sequence of SEQ ID NO:8. In embodiments, the heavy chainvariable domain is the sequence of SEQ ID NO:8. In embodiments, thelight chain variable domain includes a FR L1 as set forth in SEQ IDNO:9, a FR L2 as set forth in SEQ ID NO:10, FR L3 as set forth in SEQ IDNO:11 and a FR L4 as set forth in SEQ ID NO:12. In embodiments, theheavy chain variable domain includes a FR H1 as set forth in SEQ IDNO:13, a FR H2 as set forth in SEQ ID NO:14, FR H3 as set forth in SEQID NO:15 and a FR H4 as set forth in SEQ ID NO:16.

In embodiments, the recombinant protein (bispecific antibody) includes atryptophan at a position corresponding to Kabat position 366. Inembodiments, the recombinant protein (bispecific antibody) includes aserine at a position corresponding to Kabat position 366. Inembodiments, the recombinant protein (bispecific antibody) includes aalanine at a position corresponding to Kabat position 368. Inembodiments, the recombinant protein (bispecific antibody) includes avaline at a position corresponding to Kabat position 407. Inembodiments, the recombinant protein (bispecific antibody) includes analanine at a position corresponding to Kabat position 234. Inembodiments, the recombinant protein (bispecific antibody) includes analanine at a position corresponding to Kabat position 235.

In embodiments, the first antibody region is a first Fab′ fragment orthe second antibody region is a second Fab′ fragment. In embodiments,the first antibody region is a single chain variable fragment (scFv) orthe second antibody region is a second single chain variable fragment(scFv). In embodiments, the second scFv includes the sequence of SEQ IDNO:17. In embodiments, the second scFv is the sequence of SEQ ID NO:17.

In embodiments, the second antibody region is capable of binding IL1RAPwith an equilibrium dissociation constant (K_(D)) of about 21 nM. Inembodiments, the second antibody region is capable of binding IL1RAPwith an equilibrium dissociation constant (K_(D)) of 21 nM.

In embodiments, the second antibody region has an EC₅₀ of about 4.9ng/ml. In embodiments, the second antibody region has an EC₅₀ of 4.9ng/ml.

In embodiments, the second antibody region is bound to an IL1RAP. Inembodiments, the IL1RAP is a human IL1RAP. In embodiments, the IL1RAPforms part of a cell. In embodiments, the IL1RAP is expressed on thesurface of the cell.

In embodiments, the cell is a cancer cell. In embodiments, the cancercell is a leukemia stem cell (LSC). In embodiments, the cancer cell isan acute myeloid leukemia (AML) cell.

In one embodiment, the recombinant protein is a bispecific antibodyincluding a first antibody region of SEQ ID NO:21 and a second antibodyregion of SEQ ID NO:20.

Nucleic Acid Compositions

In an aspect, an isolated nucleic acid encoding an antibody as providedherein including embodiments thereof is provided. In embodiments, thenucleic acid includes the sequence of SEQ ID NO:18. In embodiments, thenucleic acid is the sequence of SEQ ID NO:18.

In another aspect, an isolated nucleic acid encoding a recombinantprotein as provided herein, including embodiments thereof, is provided.In embodiments, the nucleic acid includes the sequence of SEQ ID NO:18.

Pharmaceutical Compositions

In an aspect is provided a pharmaceutical composition including atherapeutically effective amount of an antibody as provided hereinincluding embodiments thereof and a pharmaceutically acceptableexcipient.

In another aspect is provided a pharmaceutical composition including atherapeutically effective amount of a recombinant protein as providedherein, including embodiments thereof, and a pharmaceutically acceptableexcipient.

Methods of Treatment

The compositions provided herein, including embodiments thereof, arecontemplated as providing effective treatments for diseases such ascancer (e.g., leukemia [e.g., AML]). Thus, in an aspect is provided amethod of treating cancer in a subject in need thereof, the methodincluding administering to a subject a therapeutically effective amountof an antibody as provided herein including embodiments thereof, therebytreating cancer in the subject.

In another aspect is provided a method of treating cancer in a subjectin need thereof, the method including administering to a subject atherapeutically effective amount of a recombinant protein as describedherein, including embodiments thereof, thereby treating cancer in thesubject. In embodiments, the cancer is leukemia. In embodiments, thecancer is acute myeloid leukemia. In embodiments, the cancer is chronicmyeloid leukemia (CML). In embodiments, the cancer is lung cancer. Inembodiments, the cancer is non-small cell lung cancer (NSCLC). Inembodiments, the cancer is pancreatic cancer. In embodiments, the canceris melanoma. In embodiments, the cancer is breast cancer. Inembodiments, the cancer is colon cancer. In embodiments, the methodfurther includes administering to the subject a second therapeuticagent.

In embodiments, the antibody is administered at an amount from about0.01 nM to about 10 nM. In embodiments, the antibody is administered atan amount from about 0.05 nM to about 10 nM. In embodiments, theantibody is administered at an amount from about 0.1 nM to about 10 nM.In embodiments, the antibody is administered at an amount from about 0.5nM to about 10 nM. In embodiments, the antibody is administered at anamount from about 1 nM to about 10 nM. In embodiments, the antibody isadministered at an amount from about 2 nM to about 10 nM. Inembodiments, the antibody is administered at an amount from about 4 nMto about 10 nM. In embodiments, the antibody is administered at anamount from about 6 nM to about 10 nM. In embodiments, the antibody isadministered at an amount from about 4 nM to about 10 nM. Inembodiments, the antibody is administered at an amount from about 8 nMto about 10 nM. In embodiments, the antibody is administered at anamount of about 0.01 nM, 0.05 nM, 0.1 nM, 0.5 nM, 1 nM, 2 nM, 2 nM, 4nM, 5 nM, 6 nM, 7 nM, 8 nM, 9 nM or 10 nM.

In embodiments, the antibody is administered at an amount from 0.01 nMto 10 nM. In embodiments, the antibody is administered at an amount from0.05 nM to 10 nM. In embodiments, the antibody is administered at anamount from 0.1 nM to 10 nM. In embodiments, the antibody isadministered at an amount from 0.5 nM to 10 nM. In embodiments, theantibody is administered at an amount from 1 nM to 10 nM. Inembodiments, the antibody is administered at an amount from 2 nM to 10nM. In embodiments, the antibody is administered at an amount from 4 nMto 10 nM. In embodiments, the antibody is administered at an amount from6 nM to 10 nM. In embodiments, the antibody is administered at an amountfrom 4 nM to 10 nM. In embodiments, the antibody is administered at anamount from 8 nM to 10 nM. In embodiments, the antibody is administeredat an amount of 0.01 nM, 0.05 nM, 0.1 nM, 0.5 nM, 1 nM, 2 nM, 2 nM, 4nM, 5 nM, 6 nM, 7 nM, 8 nM, 9 nM or 10 nM.

In embodiments, the antibody is administered at an amount from about0.01 nM to about 8 nM. In embodiments, the antibody is administered atan amount from about 0.05 nM to about 8 nM. In embodiments, the antibodyis administered at an amount from about 0.1 nM to about 8 nM. Inembodiments, the antibody is administered at an amount from about 0.5 nMto about 8 nM. In embodiments, the antibody is administered at an amountfrom about 1 nM to about 8 nM. In embodiments, the antibody isadministered at an amount from about 2 nM to about 8 nM. In embodiments,the antibody is administered at an amount from about 4 nM to about 8 nM.In embodiments, the antibody is administered at an amount from about 6nM to about 8 nM. In embodiments, the antibody is administered at anamount from about 4 nM to about 8 nM.

In embodiments, the antibody is administered at an amount from 0.01 nMto 8 nM. In embodiments, the antibody is administered at an amount from0.05 nM to 8 nM. In embodiments, the antibody is administered at anamount from 0.1 nM to 8 nM. In embodiments, the antibody is administeredat an amount from 0.5 nM to 8 nM. In embodiments, the antibody isadministered at an amount from 1 nM to 8 nM. In embodiments, theantibody is administered at an amount from 2 nM to 8 nM. In embodiments,the antibody is administered at an amount from 4 nM to 8 nM. Inembodiments, the antibody is administered at an amount from 6 nM to 8nM. In embodiments, the antibody is administered at an amount from 4 nMto 8 nM.

In embodiments, the antibody is administered at an amount from about0.01 nM to about 6 nM. In embodiments, the antibody is administered atan amount from about 0.05 nM to about 6 nM. In embodiments, the antibodyis administered at an amount from about 0.1 nM to about 6 nM. Inembodiments, the antibody is administered at an amount from about 0.5 nMto about 8 nM. In embodiments, the antibody is administered at an amountfrom about 1 nM to about 6 nM. In embodiments, the antibody isadministered at an amount from about 2 nM to about 6 nM. In embodiments,the antibody is administered at an amount from about 4 nM to about 6 nM.

In embodiments, the antibody is administered at an amount from 0.01 nMto 6 nM. In embodiments, the antibody is administered at an amount from0.05 nM to 6 nM. In embodiments, the antibody is administered at anamount from 0.1 nM to 6 nM. In embodiments, the antibody is administeredat an amount from 0.5 nM to 6 nM. In embodiments, the antibody isadministered at an amount from 1 nM to 6 nM. In embodiments, theantibody is administered at an amount from 2 nM to 6 nM. In embodiments,the antibody is administered at an amount from 4 nM to 6 nM.

In embodiments, the antibody is administered at an amount from about0.01 nM to about 4 nM. In embodiments, the antibody is administered atan amount from about 0.05 nM to about 4 nM. In embodiments, the antibodyis administered at an amount from about 0.1 nM to about 4 nM. Inembodiments, the antibody is administered at an amount from about 0.5 nMto about 4 nM. In embodiments, the antibody is administered at an amountfrom about 1 nM to about 4 nM. In embodiments, the antibody isadministered at an amount from about 2 nM to about 4 nM.

In embodiments, the antibody is administered at an amount from 0.01 nMto 4 nM. In embodiments, the antibody is administered at an amount from0.05 nM to 4 nM. In embodiments, the antibody is administered at anamount from 0.1 nM to 4 nM. In embodiments, the antibody is administeredat an amount from 0.5 nM to 4 nM. In embodiments, the antibody isadministered at an amount from 1 nM to 4 nM. In embodiments, theantibody is administered at an amount from 2 nM to 4 nM.

In embodiments, the antibody is administered at an amount from about0.01 nM to about 2 nM. In embodiments, the antibody is administered atan amount from about 0.05 nM to about 2 nM. In embodiments, the antibodyis administered at an amount from about 0.1 nM to about 2 nM. Inembodiments, the antibody is administered at an amount from about 0.5 nMto about 2 nM. In embodiments, the antibody is administered at an amountfrom about 1 nM to about 2 nM.

In embodiments, the antibody is administered at an amount from 0.01 nMto 2 nM. In embodiments, the antibody is administered at an amount from0.05 nM to 2 nM. In embodiments, the antibody is administered at anamount from 0.1 nM to 2 nM. In embodiments, the antibody is administeredat an amount from 0.5 nM to 2 nM. In embodiments, the antibody isadministered at an amount from 1 nM to 2 nM.

In embodiments, the antibody is administered at an amount from about0.01 nM to about 1 nM. In embodiments, the antibody is administered atan amount from about 0.05 nM to about 1 nM. In embodiments, the antibodyis administered at an amount from about 0.1 nM to about 1 nM. Inembodiments, the antibody is administered at an amount from about 0.5 nMto about 1 nM.

In embodiments, the antibody is administered at an amount from 0.01 nMto 1 nM. In embodiments, the antibody is administered at an amount from0.05 nM to 1 nM. In embodiments, the antibody is administered at anamount from 0.1 nM to 1 nM. In embodiments, the antibody is administeredat an amount from 0.5 nM to 1 nM.

In embodiments, the antibody is administered at an amount of about 3.15nM. In embodiments, the antibody is administered at an amount of 3.15nM. In embodiments, the antibody is administered at an amount of about1.05 nM. In embodiments, the antibody is administered at an amount of1.05 nM.

It is understood that the recombinant protein (i.e., the bispecificantibody or the chimeric antigen receptor) provided herein includingembodiments thereof may be administered at any of the concentrationsdescribed herein for the administration of the antibody (e.g., 0.01nM-10 nM).

In embodiments, the antibody is administered at an amount from about 10μg to about 500 μg. In embodiments, the antibody is administered at anamount from about 20 μg to about 500 μg. In embodiments, the antibody isadministered at an amount from about 30 μg to about 500 μg. Inembodiments, the antibody is administered at an amount from about 40 μgto about 500 μg. In embodiments, the antibody is administered at anamount from about 50 μg to about 500 μg. In embodiments, the antibody isadministered at an amount from about 60 μg to about 500 μg. Inembodiments, the antibody is administered at an amount from about 70 μgto about 500 μg. In embodiments, the antibody is administered at anamount from about 80 μg to about 500 μg. In embodiments, the antibody isadministered at an amount from about 90 μg to about 500 μg. Inembodiments, the antibody is administered at an amount from about 100 μgto about 500 μg.

In embodiments, the antibody is administered at an amount from about 110μg to about 500 μg. In embodiments, the antibody is administered at anamount from about 120 μg to about 500 μg. In embodiments, the antibodyis administered at an amount from about 130 μg to about 500 μg. Inembodiments, the antibody is administered at an amount from about 140 μgto about 500 μg. In embodiments, the antibody is administered at anamount from about 150 μg to about 500 μg. In embodiments, the antibodyis administered at an amount from about 160 μg to about 500 μg. Inembodiments, the antibody is administered at an amount from about 170 μgto about 500 μg. In embodiments, the antibody is administered at anamount from about 180 μg to about 500 μg. In embodiments, the antibodyis administered at an amount from about 190 μg to about 500 μg. Inembodiments, the antibody is administered at an amount from about 200 μgto about 500 μg.

In embodiments, the antibody is administered at an amount from about 210μg to about 500 μg. In embodiments, the antibody is administered at anamount from about 220 μg to about 500 μg. In embodiments, the antibodyis administered at an amount from about 230 μg to about 500 μg. Inembodiments, the antibody is administered at an amount from about 240 μgto about 500 μg. In embodiments, the antibody is administered at anamount from about 250 μg to about 500 μg. In embodiments, the antibodyis administered at an amount from about 260 μg to about 500 μg. Inembodiments, the antibody is administered at an amount from about 270 μgto about 500 μg. In embodiments, the antibody is administered at anamount from about 280 μg to about 500 μg. In embodiments, the antibodyis administered at an amount from about 290 μg to about 500 μg. Inembodiments, the antibody is administered at an amount from about 300 μgto about 500 μg.

In embodiments, the antibody is administered at an amount from about 310μg to about 500 μg. In embodiments, the antibody is administered at anamount from about 320 μg to about 500 μg. In embodiments, the antibodyis administered at an amount from about 330 μg to about 500 μg. Inembodiments, the antibody is administered at an amount from about 340 μgto about 500 μg. In embodiments, the antibody is administered at anamount from about 350 μg to about 500 μg. In embodiments, the antibodyis administered at an amount from about 360 μg to about 500 μg. Inembodiments, the antibody is administered at an amount from about 370 μgto about 500 μg. In embodiments, the antibody is administered at anamount from about 380 μg to about 500 μg. In embodiments, the antibodyis administered at an amount from about 390 μg to about 500 μg. Inembodiments, the antibody is administered at an amount from about 400 μgto about 500 μg.

In embodiments, the antibody is administered at an amount from about 410μg to about 500 μg. In embodiments, the antibody is administered at anamount from about 420 μg to about 500 μg. In embodiments, the antibodyis administered at an amount from about 430 μg to about 500 μg. Inembodiments, the antibody is administered at an amount from about 440 μgto about 500 μg. In embodiments, the antibody is administered at anamount from about 450 μg to about 500 μg. In embodiments, the antibodyis administered at an amount from about 460 μg to about 500 μg. Inembodiments, the antibody is administered at an amount from about 470 μgto about 500 μg. In embodiments, the antibody is administered at anamount from about 480 μg to about 500 μg. In embodiments, the antibodyis administered at an amount from about 490 μg to about 500 μg.

In embodiments, the antibody is administered at an amount from about 10μg to about 400 μg. In embodiments, the antibody is administered at anamount from about 20 μg to about 400 μg. In embodiments, the antibody isadministered at an amount from about 30 μg to about 400 μg. Inembodiments, the antibody is administered at an amount from about 40 μgto about 400 μg. In embodiments, the antibody is administered at anamount from about 50 μg to about 400 μg. In embodiments, the antibody isadministered at an amount from about 60 μg to about 400 μg. Inembodiments, the antibody is administered at an amount from about 70 μgto about 400 μg. In embodiments, the antibody is administered at anamount from about 80 μg to about 400 μg. In embodiments, the antibody isadministered at an amount from about 90 μg to about 400 μg. Inembodiments, the antibody is administered at an amount from about 100 μgto about 400 μg.

In embodiments, the antibody is administered at an amount from about 10μg to about 300 μg. In embodiments, the antibody is administered at anamount from about 20 μg to about 300 μg. In embodiments, the antibody isadministered at an amount from about 30 μg to about 300 μg. Inembodiments, the antibody is administered at an amount from about 40 μgto about 300 μg. In embodiments, the antibody is administered at anamount from about 50 μg to about 300 μg. In embodiments, the antibody isadministered at an amount from about 60 μg to about 300 μg. Inembodiments, the antibody is administered at an amount from about 70 μgto about 300 μg. In embodiments, the antibody is administered at anamount from about 80 μg to about 300 μg. In embodiments, the antibody isadministered at an amount from about 90 μg to about 300 μg. Inembodiments, the antibody is administered at an amount from about 100 μgto about 300 μg.

In embodiments, the antibody is administered at an amount from about 10μg to about 200 μg. In embodiments, the antibody is administered at anamount from about 20 μg to about 200 μg. In embodiments, the antibody isadministered at an amount from about 30 μg to about 200 μg. Inembodiments, the antibody is administered at an amount from about 40 μgto about 200 μg. In embodiments, the antibody is administered at anamount from about 50 μg to about 200 μg. In embodiments, the antibody isadministered at an amount from about 60 μg to about 200 μg. Inembodiments, the antibody is administered at an amount from about 70 μgto about 200 μg. In embodiments, the antibody is administered at anamount from about 80 μg to about 200 μg. In embodiments, the antibody isadministered at an amount from about 90 μg to about 200 μg. Inembodiments, the antibody is administered at an amount from about 100 μgto about 200 μg.

In embodiments, the antibody is administered at an amount from about 10μg to about 100 μg. In embodiments, the antibody is administered at anamount from about 20 μg to about 100 μg. In embodiments, the antibody isadministered at an amount from about 30 μg to about 100 μg. Inembodiments, the antibody is administered at an amount from about 40 μgto about 100 μg. In embodiments, the antibody is administered at anamount from about 50 μg to about 100 μg. In embodiments, the antibody isadministered at an amount from about 60 μg to about 100 μg. Inembodiments, the antibody is administered at an amount from about 70 μgto about 100 μg. In embodiments, the antibody is administered at anamount from about 80 μg to about 100 μg. In embodiments, the antibody isadministered at an amount from about 90 μg to about 100 μg.

In embodiments, the antibody is administered at an amount of about 10μg, 20 μg, 30 μg, 40 μg, 50 μg, 60 μg, 70 μg, 80 μg, 90 μg, 100 μg, 110μg, 120 μg, 130 μg, 140 μg, 150 μg, 160 μg, 170 μg, 180 μg, 190 μg, 200μg, 210 μg, 220 μg, 230 μg, 240 μg, 250 μg, 260 μg, 270 μg, 280 μg, 290μg, 300 μg, 310 μg, 320 μg, 330 μg, 340 μg, 350 μg, 360 μg, 370 g, 380μg, 390 μg, 400 μg, 410 μg, 420 μg, 430 μg, 440 μg, 450 μg, 460 μg, 470μg, 480 μg, 490 μg, or 500 μg.

In embodiments, the antibody is administered at an amount of 10 μg, 20μg, 30 μg, 40 μg, 50 g, 60 μg, 70 μg, 80 μg, 90 μg, 100 μg, 110 μg, 120μg, 130 μg, 140 μg, 150 μg, 160 μg, 170 μg, 180 μg, 190 μg, 200 μg, 210μg, 220 μg, 230 μg, 240 μg, 250 μg, 260 g, 270 μg, 280 μg, 290 μg, 300μg, 310 μg, 320 μg, 330 μg, 340 μg, 350 μg, 360 μg, 370 g, 380 μg, 390μg, 400 μg, 410 μg, 420 μg, 430 g, 440 μg, 450 μg, 460 g, 470 μg, 480μg, 490 μg, or 500 μg.

It is understood that the recombinant protein (i.e., the bispecificantibody or the chimeric antigen receptor) provided herein includingembodiments thereof may be administered at any of the concentrationsdescribed herein for the administration of the antibody (e.g., 10 μg-500μg).

In embodiments, the recombinant protein or antibody is administered atan amount of about 200 μg. In embodiments, the recombinant protein orantibody is administered at an amount of 200 μg.

Methods of Inhibiting Cell Proliferation

The compositions provided herein, including embodiments thereof, arefurther contemplated for inhibiting cell proliferation. Thus, in anaspect is provided a method of inhibiting proliferation of a cell, themethod including: (i) contacting a cell with an anti-IL1RAP antibody asprovided herein including embodiments thereof, or a recombinant proteinas provided herein including embodiments thereof, thereby forming acontacted cell; and (ii) allowing the anti-IL1RAP antibody, therecombinant protein as provided herein including embodiments thereof tobind an IL1RAP on the contacted cell, thereby inhibiting proliferationof the cell. In embodiments, the cell is a cancer cell. In embodiments,the cell is a leukemia stem cell (LSC). In embodiments, the cancer cellis an acute myeloid leukemia (AML) cell. In embodiments, the cancer cellis a chronic myeloid leukemia (CML) cell. In embodiments, the cancercell is a lung cancer cell. In embodiments, the cancer cell is anon-small cell lung cancer (NSCLC) cell. In embodiments, the cancer cellis a pancreatic cancer cell. In embodiments, the cancer cell is amelanoma cell. In embodiments, the cancer cell is a breast cancer cell.In embodiments, the cancer cell is a colon cancer cell.

EXAMPLES

The following examples are offered to illustrate, but not to limit theclaimed invention.

Example 1

Acute myeloid leukemia (AML) is associated with a poor survival rate,and there is an urgent need for novel and more efficient therapiesideally targeting AML stem cells that are essential for maintaining thedisease. Applicants' lab and other publications reported that IL1RAPprotein is upregulated on candidate leukemic stem cells in the majorityof AML patients, but not on normal hematopoietic stem cells.

Applicants developed a new human monoclonal antibody (1D5) that can bindIL1RAP on AML cells and applied this antibody for bispecific antibody(anti-CD3x IL1RAP) and CAR-T techniques. Applicants' study showed thatthe bispecific antibody can efficiently kill all AML cell lines testedand ten-patient AML sample as well. The in vivo study also demonstratesits therapeutic potency.

Both the in vitro and in vivo experimental results show that theantibody possesses great potential for treating AML disease in theclinic.

Applicants screened a monoclonal antibody (1D5) from their human naïvephage display antibody library against IL1RAP protein which is reportedto be upregulated in Acute Myeloid Leukemia (AML) cells. This antibodyshowed affinity of around 20 nM to recombinant IL1RAP protein.

Using this monoclonal antibody (1D5), Applicants then synthesized abispecific antibody. The bispecific antibody consists of two arms: oneis CD3 antibody, and the other one is the monoclonal antibody 1D5. Thisbispecific antibody binds to cells that express IL1RAP protein, and thenhelps to recruit T cell to the IL1RAP-expressing cells and kills thecells. Applicants next tested the binding of this antibody to sevendifferent AML cell lines. After confirmation of the binding of thisantibody to all AML cells lines tested, Applicants tried a specifickilling assay with all 7 AML cell lines and 10 AML patient samples. Thekilling assay showed over 90% of specific killing for both AML celllines and AML patient sample.

Applicants' in vivo study also demonstrated that by infusing thisbispecific antibody in mice that are engrafted with MV4-11 cell linetogether with human T cells, the MV4-11 cells are eliminated compared tothe control group.

This bispecific antibody can be further developed for the treatment ofAML. The monoclonal antibody 1D5 is now used for CAR-T which is anotherpowerful approach to treat AML.

Example 2

Immunotherapeutic targeting of IL1RAP to eliminate leukemia stem cells(LSC) in Acute Myeloid Leukemia (AML).

Abstract. AML is a devastating hematopoietic malignancy, resulting inprogressive accumulation of primitive and partially differentiatedclonal blasts and rapidly leads to hematopoiesis failure and death ifleft untreated. Despite increasing knowledge of the molecular mechanismsof leukemogenesis, implementation of risk-stratification strategies fortreatment guidance and use of molecular targeting therapeutics, only aminority of AML patients are cured with current chemotherapy regimenswhile the majority of them are either immediately refractory toinduction therapy or subsequently relapse following transient completeremission. The current view is that treatment resistance and relapse inAML is due to the inability of chemotherapy and/or other moleculartargeting therapeutics to eliminate the so-called leukemia stem cells(LSC). These clonal primitive cells are at the hierarchical apex ofleukemogenesis and have stem cell properties to maintain clonalexpansion indefinitely. Applicants' goal is to develop novel therapiesthat can effectively eradicate LSC and improve cure rates in AMLpatients, while sparing normal hematopoietic stem cells (HSC).

Allogeneic hematopoietic cell transplantation (alloHCT) has beensuccessfully applied in AML patients and has provided the proof ofconcept that a normally functioning (donor) immune system can target andeliminate LSC. However, because intrinsic toxicity andtransplant-related mortality, alloHCT can be performed only in subsetsof AML patients. More recently, other immunotherapeutic approaches, suchas naked, conjugated or T cell engaged bispecific monoclonal antibodies(BsAb) recruiting T or NK cells or chimeric antigen receptor (CAR)engineered T cells, have been shown to be a feasible and effectivetreatment approach in cancer and leukemia with relatively minortoxicity. BsAb or CAR T cells redirect the immune system to specificantigen targets expressed on the surface of cancer or leukemia cells,thereby enhancing immune-mediated specific killing of malignant cells.Here Applicants propose to select novel membrane antigens that arespecifically enriched on AML LSC (compared to normal HSC) and targetthem using T cell engaged BsAb and/or CAR T-cells. Recently Applicantshave focused on Interleukin-1 receptor accessory protein (IL1RAP).Overactivation of innate immune components such as TLRs, IRAK/TRAF6,IL8/CXCR2 and IL1 signaling pathways has been demonstrated in LSC.IL1RAP is a coreceptor of IL1 receptor. Applicants have shown thatIL1RAP is selectively upregulated on chronic myeloid leukemia (CML) LSCcompared to normal HSC, and blocking IL-1 signaling with IL-1 receptorantagonist (IL-iRA) inhibited leukemia growth, while preserving normalhematopoiesis. Applicants found that IL1RAP is also over-expressed onthe surface of human AML CD34⁺ blasts compared with normal CD34⁺ cells,and others have shown that high IL1RAP expression is associated withpoor overall survival in AML. Altogether, these data suggest that IL1RAPis an ideal target for targeting LSC.

Applicants have utilized a human naïve phage display library made from10 healthy donors to develop functional IL1RAP monoclonal and T cellengaged BsAb and CAR-T cells. Preliminary data indicated that theconstruct based on IL1RAP epitopes derived from the aforementioned humannaïve phage display library can be utilized to produce high affinityBsAb and CAR-T cells. As proof of principle, Applicants showed specifickilling of both AML cell lines and primary AML patient leukemia blastsin the T cell Dependent Cellular Cytotoxicity (TDCC) assay in thepresence of anti-IL1RAPXCD3 BsAb in vitro, and disease elimination inimmunodeficient mice engrafted with MV4-11 AML cells and treated withboth BsAb and human T-cells in vivo.

Thus, Applicants have a novel construct to produce immunotherapeuticstargeting IL1RAP on AML LSC. Applicants will optimize the therapeuticefficacy, dose and schedule of anti-IL1RAPxCD3 BsAb to target LSC inAML, develop IL1RAP CAR-T cells to eradicate human AML LSC and determineclinical applicability, and enhance anti-leukemic activity ofIL1RAP-based immunotherapeutics using immunocheckpoint inhibitors. Forthese experiments, Applicants will utilize multiple AML mouse models andsequential transplants of primary AML blasts in immunodeficient mice tofunctionally demonstrate elimination of LSC.

Background Acute myeloid leukemia (AML) is a devastating hematopoieticmalignancy, resulting in progressive accumulation of primitive andpartially-differentiated clonal blasts, a condition rapidly leading tohematopoiesis failure and death if untreated [1]. AML blasts arecharacterized by specific molecular profiles in individual patients,including cytogenetic aberrations, gene mutations, aberrant coding andnon-coding gene RNA levels and epigenetic changes. This heterogeneity isclinically relevant and has been used to select the most appropriaterisk-adapted treatments. However, despite increasing knowledge ofmolecular mechanisms of leukemogenesis and implementation ofrisk-stratification treatment strategies, only a minority of the AMLpatients are cured, while the vast majority of them are eitherimmediately refractory to chemotherapy or relapse following transientachievement of complete remission[2, 3]. The inability of chemotherapyand/or other molecular targeting therapeutics to cure AML is likely dueto persistence of the so-called leukemia stem cells (LSC). These clonalprimitive cells are at the hierarchical apex of the leukemogenic processand have stem cell properties that maintain clonal expansionindefinitely. Thus, Applicants' goal is to develop novel therapies thateffectively eradicate LSC and improve cure rates in AML patients, whilesparing normal hematopoietic stem cells (HSC). Allogeneic hematopoieticcell transplantation (alloHCT) has been successfully applied to AMLpatients and has provided the proof of concept that a normallyfunctioning (donor) immune system can target and eliminate LSC [4, 5].However, because of transplant-related toxicity and mortality, alloHCTis feasible only in subsets of AML patients. More recently, therapiesthat harness the immune system like naked, conjugated or T cell-engagedbispecific monoclonal antibodies (BsAb), immunocheckpoint inhibitors orchimeric antigen receptor (CAR) engineered T cells have been shown to befeasible and effective in advanced human malignancies, including solidtumors, lymphomas and acute leukemias. Of these approaches, BsAb orCAR-T cells are both based on the principle of redirecting the immunesystem to specific antigen targets on cancer cells with little toxicity[6, 7]. Applicants propose to select novel membrane antigensspecifically enriched on AML LSC (compared to normal HSC) and targetthem using BsAb and/or CAR-T cells. Recently, Applicants have focused onInterleukin-1 receptor accessory protein (IL1RAP), an IL1R co-receptor,given that overactivation of innate immune components such as TLRs,IRAK/TRAF6, IL8/CXCR2 and IL1 signaling pathways is reportedly essentialfor LSC homeostasis. Applicants also found that IL1RAP is over-expressedon the surface of human AML CD34⁺blasts compared with normal CD34⁺cells. Similar results were reported by Barreyo et al which show thathigh levels of IL1RAP protein are expressed on the surface of HSC frompatients with AML and high-risk myelodysplastic syndrome, and areassociated with poor overall survival [9].

Methods.

Optimize therapeutic efficacy, dose and schedule of anti-IL1RAPxCD3 BsAbto target AML LSC. T cell-redirected tumor cell killing is a promisingimmunologic approach to treat hematologic malignancies [7, 10, 11]. BsAbcan redirect polyclonal T cells against tumor cells by binding to tumorantigen and T cell co-receptor CD3. This interaction induces activationand cytotoxicity of T effector cells against target cancer cells in amajor histocompatibility complex-independent manner, bypassing an immuneescape mechanism [7]. To create anti-IL1RAPxCD3 BsAb, Applicants firstconstructed a human phage display library from healthy donor-derivedperipheral blood B cells (FIGS. 1A-1C, Table 1) and screened 12 clonesproducing anti-IL1RAP monoclonal antibodies (Ab). Ab from clone ID5showed the highest binding ability to recombinant human IL1RAP proteinusing ELISA (FIGS. 2A-2B, 2D and Table 2, with an EC₅₀ of 4.9 ng/ml) andthe highest specific lysis rate in Antibody Dependent CellularCytotoxicity (ADCC) assays (FIG. 2C), and therefore was selected fordevelopment of anti-IL1RAPxCD3 BsAb (FIGS. 3A-3C) and IL1RAP-CAR-T cells(FIGS. 7A-7B). The EC₅₀ values determined in the 48 hours long termkilling assays with different cell lines are: 6.718 ng/ml for HL60cells, 5.362 ng/ml for MOLM13 cells, 6.368 ng/ml for OCI-AML3 cells,

TABLE 1 List of CDR3H and CDR3L amino acid sequences from 50randomly selected clones. The CDR3 sequences showedthat no repetitive sequences could be found fromthese randomly picked 50 colonies. CDR3H-IMGT CDR3L-IMGTARDESHSTITPRH (SEQ ID NO: 22) QCGIWVIIFM (SEQ ID NO: 56)ARVGPHYSDSSGYYYIDDNSYDMDV (SEQ ID LQHRSGYL (SEQ ID NO: 57) NO: 23)ARDVWWPAAPLYYYYGMDV (SEQ ID NO: 24) MQGIHLPRYT (SEQ ID NO: 58)ARDGDYGVWWFDP (SEQ ID NO: 25) QQYYSSPLT (SEQ ID NO: 59)ARGRLASGSWNGFDI (SEQ. ID NO: 26) QQYYSMVSLT (SEQ ID NO: 60)AAESGRPGFGSYWGVFYYNHAMDV (SEQ ID LVWHNRAWV (SEQ ID NO: 61) NO: 27)ARDGNVPMASDFYGMDL (SEQ ID NO: 28) QQYDNLLLT (SEQ ID NO: 62)ARGKGDSYAFDI (SEQ ID NO: 29) QAWDNTSQYV (SEQ ID NO: 63)AKGHYYYYGMDV (SEQ ID NO: 30) QQSYTVPYT (SEQ ID NO: 64)ARAGSGWYGYFDS (SEQ ID NO: 31) QKYNSAPYT (SEQ ID NO: 65)ARDSERVVSGWYVYYYYYYMDV (SEQ ID QVWDRSGDHQGV (SEQ ID NO: 66) NO: 32)AKDLRGYSYGNYNRDAFDI (SEQ ID NO: 33) QSYDSSLSGSGVV (SEQ ID NO: 67)ARGKGDYLYYGMDV (SEQ ID NO: 34) QYYNTYSPWT (SEQ ID NO: 68)ARPEGGSSLVGGFDY (SEQ ID NO: 35) AVWDDSLKGVV (SEQ ID NO: 69)ARFGGATFDGPFDI (SEQ ID NO: 36) LQHNTYPWT (SEQ ID NO: 70)ARRDTAMENFDY (SEQ ID NO: 37) QQRSNWPELT (SEQ ID NO: 71)ARLYYDFWSGSA (SEQ ID NO: 38) MQALQTPWT (SEQ ID NO: 72)ARRSPDCSLTTCLPLH (SEQ ID NO: 39) QQYADSPLT (SEQ ID NO: 73)VRDVSPGGADV (SEQ ID NO: 40) QQYGRSPPFA (SEQ ID NO: 74)AKGGTLDAFGI (SEQ ID NO: 41) QQSHSSSR (SEQ ID NO: 75)ARAFNRYCSGGSCYPPGRYGMDV (SEQ ID CSYAGAYTEI (SEQ ID NO: 76) NO: 42)ARDHESRSPGDYHMDV (SEQ ID NO: 43) HQYGSIPHT (SEQ ID NO: 77)ASLDLTAARSVIAAFDI (SEQ ID NO: 44) GADHGSGSNFLYV (SEQ ID NO: 78)ARGDSSDYWTSTDAFDI (SEQ ID NO: 45) QVWDSGSDQGV (SEQ ID NO: 79)AIPYGDYPPAFAV (SEQ ID NO: 46) QQYHTIPYS (SEQ ID NO: 80)ARDSPEGAFDI (SEQ ID NO: 47) QQYGSSPRT (SEQ ID NO: 81)ARFNCYSSGCPLMDY (SEQ ID NO: 48) LQYGRSPFT (SEQ ID NO: 82)ARDSGSYLFDY (SEQ ID NO: 49) CSMGCHPKCLWA (SEQ ID NO: 83)ARTLPYDFWSGYSAYYYYYYYMDV (SEQ ID MQGLQTPIG (SEQ ID NO: 84) NO: 50)AREDGHTGIYDY (SEQ ID NO: 51) CSYVGRYTYV (SEQ ID NO: 85)ARGAEYSSSSPDY (SEQ ID NO: 52) QQYNTYPRA (SEQ ID NO: 86)ARQGNIVVVVANDAFDI (SEQ ID NO: 53) QHYGSSLWP (SEQ ID NO: 87)AKEVRPGHCSGGSGGSCYSVPGRDYYGMDV MQALQTPVT (SEQ ID NO: 88) (SEQ ID NO: 54)AGPSGPAKKDAFDI (SEQ ID NO: 55) QRYNNWPPGIT (SEQ ID NO: 89)

TABLE 2 Biopanning for IL1RAP antibodies using recombinant human IL1RAPprotein. Enrichment was observed at the fourth round. See alsocorresponding FIG. 1D. 1^(st) round 2^(nd) round 3^(rd) round 4^(th)round Input  1 × 10¹³  1 × 10¹²  1 × 10¹²  1 × 10¹² Output 1 × 10⁸ 3.5 ×10⁵   8.1 × 10⁵   4.6 × 10⁷   Output/input ratio 10⁻⁵ 3.5 × 10⁻⁷ 8.1 ×10⁻⁷ 4.6 × 10⁻⁵

Evaluate correlation between efficacy of anti-IL1RAPxCD3 BsAb-induced Tcell killing and IL1RAP expression levels in primary blasts from subsetsof AML patients. Applicants showed that IL1RAP is expressed on thesurface of AML cell lines (FIGS. 4A-4B) and primary patient blasts(FIGS. 5A-5B), and that Applicants' anti-IL1RAPxCD3 BsAb induces T cellactivation and cytotoxicity in AMVL cell lines (FIGS. 4C-4I) and in AMVLpatient samples (FIGS. 5C-5D), with killing efficiency related to IL1RAPlevels on the surface of target cells. To obtain efficacy data based ondifferent cytogenetic and molecular subsets of AML patients, Applicantspropose to perform T cell Dependent Cellular Cytotoxicity (TDCC) assayswith anti-IL1RAPxCD3 BsAb on 30 additional patient samplesrepresentative of the three ELN 2017 cytogenetic/molecular risk groups(favorable, intermediate and adverse) [3]. Unsorted mononuclear cells(MNC) and sorted CD34⁺CD38⁺ committed progenitors and CD34⁺CD38⁻primitive progenitors from these 30 AML patient samples will beevaluated for IL1RAP expression by flow cytometry before exposing toanti-IL1RAPxCD3 BsAb and T cells. Since T cells may lose specific T cellmarkers in culture, Applicants will label AML cells with CFSE beforeco-culturing them with T cells to separate both cell types. Based onpreliminary data, Applicants will use an effector T cell to target AMLcell (E:T) ratio of 5:1 for long-term killing assays. Ab in three-foldserial dilutions starting at 330 ng/ml will be tested, in triplicate.Applicants will record counts of living target cells (DAPI⁻CSFE⁺) and Tcells, and calculate the lysis rate: % Specific lysis=(Targetcells^(+T cells)−Target cells^(+BsAb+T cells))]/Targetcells^(+T cells))×100%. EC₅₀ of BsAb for 30 AML samples will becalculated by Graphpad prism. EC₅₀ and IL1RAP expression levels will becorrelated. Enrichment of IL1RAP in specific subsets of AML patients andefficacy of the BsAb in these subsets may re-direct efforts to developthis novel drug to target specific cytogenetic/molecular AML subsets andprovide insight into employing IL1RAP as a predictive biomarker forresponse to BsAb.

Optimize dose and schedule of administration of anti-IL1RAPXCD3 BsAb andT cells to target AML LSC in vivo. Applicants showed thatanti-IL1RAPXCD3 BsAb combined with human T cells effectively eliminateleukemia burden in immunodeficient (NSG) mice transplanted with fireflyluciferase-expressing human AML cell line MV4-11 (MV4-11^(Luci)) cells(FIGS. 6A-6B). Applicants propose to conduct pharmacokinetics (PK) andpharmacodynamics (PD) studies and PK-PD modeling to allow optimizationof Ab dose/schedule. NSG mice will be treated at 3 log-dose levels (1mg/kg, 3 mg/kg, 10 mg/kg) of BsAb. Blood will be collected at 1, 2, 4,8, 24, 48, 72, 96, 120, and 148 hours (n=6 per time points×10 timepoints×3 doses=180 mice). Based on extensive experience conductingpreclinical mouse PK studies, 5-7 mice per time point provide adequateassessment of variability and characterization of PK and PD across theevaluated 1 log-fold dose range. BsAb serum concentration will bedetermined using the TDCC assay and calculated based on a standard curveobtained by serial dilution of purified BsAb. The optimal dose/scheduleof administration will be determined based on BsAb blood half-life.Next, Applicants will transplant NSG mice with 1×10⁶ MV4-11^(Luci) cellsand treat the mice with the optimized dose/schedule of BsAb and 3 doses(i.e., 1×10⁶, 3×10⁶ or 5×10⁶) of T cells at day 7 post-transplant. Invivo imaging will be taken weekly to monitor tumor burden. Calculatedtotal bioluminescence intensity levels in each mouse will represent themain PD endpoint. Relationships between PK and PD data will be initiallyevaluated using standard linear correlation and linear regression, and aPK-PD model will be built and used to simulate plasma concentration-timeprofiles and achievement of the correspondent PD endpoint to guidefuture in vivo experiments. Upon successful computer simulation of themodel, Applicants will attain one or more BsAb dose/schedule regimenspredicted to maximally decrease of bioluminescence. Applicants willselect a regimen for in vivo validation based on the following criteria:a.) lack of toxicity, b.) achievement of maximal bioluminescence change,c.) requirement for less frequent dosing, and d.) lower cost. Validationwill be performed by transplanting AML blasts from three patients intoNSG mice (16 mice/group×6 groups×2 samples=192 mice) and evaluating invivo anti-leukemic efficacy of anti-IL1RAPxCD3 BsAb and human T cells atdoses predicted by Applicants' PK/PD models. A set of mice will befollowed for survival while another set will be euthanized and BM andspleen MNC utilized in secondary transplantation experiments todetermine the treatment impact on the residual LSC burden.

Development of IL1RAP CAR-T cells to eradicate human AML LSC and todetermine their clinical applicability. T cells redirected to specificantigen targets with engineered chimeric antigen receptors (CAR) are nowa powerful therapeutic approach in hematologic malignancies [12, 13]. Toenhance reactivity, T cells are genetically modified using engineeredreceptor constructs that allow for specific targeting of tumor cellsurface antigens otherwise not (or less) immunogenic in unmodified Tcells. Applicants propose to pursue production and testing of IL1RAPCAR-T cells. Of note, tumor-targeting T cells must persist for asufficient period of time in vivo for successful tumor elimination;however, mature CAR-T cells rapidly differentiate into short-livedeffector cells, which limit their anti-tumor activity in vivo [6, 14,15]. Thus, Applicants will test both mature T cells and T cellprecursors generated from cord blood CD34⁺ cells to produce IL1RAP CAR-Tcells.

Development of IL1RAP CAR-T cells. Applicants have developed lentiviralvectors encoding IL1RAP CAR [17] (FIG. 7A). Jurkat cells transduced withIL1RAP CAR lentivirus showed cell activation compared to control cells(FIG. 7B), thereby confirming effect of IL1RAP CAR construct. Applicantspropose to purify T cells from peripheral blood mononuclear cells (PBMC)of healthy donors stimulated with OKT3/CD28 beads and interleukin-2(IL-2) at 50 IU/ml and transduce them with IL1RAP CAR lentivirus asdescribed [17]. Applicants will also generate CAR-T cell precursors asfollows. Cord blood CD34+ cells will be transduced with lentiviralvectors encoding IL1RAP CAR and cultured with immobilized Notch ligandDelta-like 4 (DL4) to promote T cell differentiation. IL1RAP CARexpression in T cells will be confirmed by detecting IL1RAP-scFv viaflow cytometry.

Efficacy evaluation of IL1RAP CAR-T cells. After obtaining mature andprecursor IL1RAP CAR-T cells, Applicants will test their activity.First, Applicants will incubate IL1RAP CAR-T cells with AML cell lines(HL60, MV4-11, THP-1, KG-1a) and Raji cells (negative control). CD107a,IFN-γ and TNF-α levels will be monitored 6 hours later by flow cytometryas an indicator of T cell activation. Killing efficiency of target AMLcells will be determined by a 4-hour ⁵¹Cr release assay. Next, IL1RAPCAR-T cells will be incubated with primary human AML cells, and T cellactivation and AML cell killing will be evaluated. For in vivo killingefficacy, MV4-11^(Luci) cells (1×10⁶/per mouse) will be transplantedinto NSG mice, and 7 days later, mice will receive single, double (days1 and 14), or triple (days 1, 14, 28) injections of 3 doses (i.e.,1×10⁶, 3×10⁶ or 5×10⁶) of IL1RAP CAR-T cells. In vivo imaging will occurweekly to monitor tumor burden until all control mice die. As described,experiments with BsAb PK-PD modeling will be performed based on bloodconcentration of IL1RAP CAR-T cells overtime and changes inbioluminescence as leukemia burden to determine the optimal dose andfrequency of IL1RAP CAR-T cells administration. Applicants will thenperform similar experiments using AML patient samples and conduct invivo efficacy, T cell persistence, and memory T cell formation andchallenging studies. Serial transplant experiments, as described abovefor the BsAb study, will also be performed to ensure LSC elimination.

Enhancement of anti-leukemic activity of IL1RAP-based immunotherapeutics(i.e., anti-IL1RAPxCD3 BsAb or IL1RAP CAR-T cells) usingimmunocheckpoint inhibitors. Despite encouraging results in preclinicalmodels and in patients, activation of immune-suppressive pathways canantagonize adoptive T-cell therapy [18]. The programmed death-1 (PD-1)pathway has emerged as a promising target for cancer therapy. PD-1 bindsto ligands PD-L1 (the predominant immunosuppression mediator upregulatedon many tumor types) and PD-L2 expressed on macrophages and dendriticcells [19] [20, 21]. Recent trials using a human-derived IgG4 PD-1monoclonal Ab (mAb; BMS-936558) show that the Ab inhibits the PD1immunocheckpoint and restores tumor cell immunogenicity, resulting indurable clinical responses in patients with advanced malignancies [19,21-24]. Herein we hypothesize that PD-1 inhibitors could enhanceadoptive T cell function when used in combination with anti-IL1RAPxCD3BsAb or IL1RAP CAR-T cells.

Determine the anti-leukemic activity of PD-1 blocking in AML cell linesand AML patient samples. T cell stimulation reportedly enhances PD-L1(B7-H1) expression on AML cell lines, and IFN-γ treatment significantlyenhances PD-L1 expression in AML patient samples [25]. Enhanced PD-L1expression induced by IFN-γ or TLR ligands, either as an immune responseto leukemia cells or released by infectious microorganisms, may protectleukemic cells against T cells. To determine whether CAR or BsAb-inducedT cell stimulation increases PD-1 expression, Applicants will employ twoways to evaluate the effect of blocking PD-1 pathway on augmenting thetherapeutic efficacy of BsAb+T cells or IL1RAP CAR-T cells treatment. Tcells will be co-cultured with MV4-11 AML cells (at E:T ratios of 1:1,1:5, and 1:10) treated with and without IFN-γ to up-regulate PD-1ligands, with and without PD-1 blocking antibody (pembrolizumab ornivolumab). The effect of PD-1 blockade on T cell cytolytic activitywill be determined based on the lysis rate. Effector cytokine secretion(IL-2, IFN-γ, and TNF-alpha) will be determined by ELISA assay. PD-1blockade will also be assessed in NSG mice engrafted with MV4-1 Luci andtreated with CAR-T, with or without PD-1 blocking antibody. Leukemiaburden will be determined by in vivo imaging and blood CAR-T cellsmeasured by flow cytometry.

Determine the anti-leukemic activity of BsAb+T cells or IL1RAP CAR-Tcells in combination with PD-1 inhibitors. If PD-1/PD-L1 blockadeenhances AML cell killing, Applicants will evaluate the effect of BsAb+Tcells or IL1RAP CAR-T cells combined with PD-1 blocking in vivo.Applicants will treat NSG mice transplanted with MV4-11Luci or human AMLpatient samples with anti-IL1RAPXCD3 BsAb and human T cells, or IL1RAPCAR-T cells, in combination with pembrolizumab or nivolumab or isotypecontrol antibodies (The basic protocol is described above). Tumor burdenin PB, BM and spleen and survival will be determined, and a 2^(nd)transplantation will be performed to monitor LSC eradication.

Statistical Considerations. Based on previous experience, to attain atleast 90% power to detect differences in survival, 11 mice per group arerequired using a one-sided test at α=0.05. Differences betweenKaplan-Meier survival curves will be assessed by the log-rank test.Descriptive and graphical statistics will be used to characterize theeffect of BsAb+T cells and IL1RAP CAR-T cells in other in vitro and PDstudies; formal comparisons will be done using t-tests and/orMann-Whitney U tests. Similar methods will be used to analyze functionalassay data.

REFERENCES

-   1. Khwaja, A., et al., Acute myeloid leukaemia. Nat Rev Dis    Primers, 2016. 2: p. 16010.-   2. Cancer Genome Atlas Research, N., et al., Genomic and epigenomic    landscapes of adult de novo acute myeloid leukemia. N Engl J    Med, 2013. 368(22): p. 2059-74.-   3. Dohner, H., et al., Diagnosis and management of AML in adults:    2017 ELN recommendationsfrom an international expert panel.    Blood, 2017. 129(4): p. 424-447.-   4. Gyurkocza, B., A. Rezvani, and R. F. Storb, Allogeneic    hematopoietic cell transplantation: the state of the art. Expert Rev    Hematol, 2010. 3(3): p. 285-99.-   5. Ringden, O. and K. Le Blanc, Allogeneic hematopoietic stem cell    transplantation: state of the art and new perspectives. APMIS, 2005.    113(11-12): p. 813-30.-   6. Kenderian, S. S., et al., Chimeric antigen receptor T-cell    therapy to target hematologic malignancies. Cancer Res, 2014.    74(22): p. 6383-9.-   7. Huehls, A. M., T. A. Coupet, and C. L. Sentman, Bispecific T-cell    engagersfor cancer immunotherapy. Immunol Cell Biol, 2015. 93(3): p.    290-6.-   8. Zhang, B., et al., Inhibition of interleukin-1 signaling enhances    elimination of tyrosine kinase inhibitor-treated CML stem cells.    Blood, 2016. 128(23): p. 2671-2682.-   9. Barreyro, L., et al., Overexpression of IL-1 receptor accessory    protein in stem and progenitor cells and outcome correlation in AML    and MDS. Blood, 2012. 120(6): p. 1290-8.-   10. Wu, J., et al., Blinatumomab: a bispecific T cell engager (BiTE)    antibody against CD19 CD3 for refractory acute lymphoid leukemia. J    Hematol Oncol, 2015. 8: p. 104.-   11. Walter, R. B., Biting back: BiTE antibodies as a promising    therapy for acute myeloid leukemia. Expert Rev Hematol, 2014.    7(3): p. 317-9.-   12. Grupp, S. A., et al., Chimeric antigen receptor-modified T cells    for acute lymphoid leukemia. N Engl J Med, 2013. 368(16): p.    1509-18.-   13. Maus, M. V., et al., Antibody-modified T cells: CARs take the    front seat for hematologic malignancies. Blood, 2014. 123(17): p.    2625-35.-   14. Dolnikov, A., et al., Antileukemic potency of CD19-specific T    cells against chemoresistant pediatric acute lymphoblastic leukemia.    Exp Hematol, 2015. 43(12): p. 1001-1014 e5.-   15. Gill, S. and C. H. June, Going viral: chimeric antigen receptor    T-cell therapy for hematological malignancies. Immunol Rev, 2015.    263(1): p. 68-89.-   16. Zakrzewski, J. L., et al., Tumor immunotherapy across MHC    barriers using allogeneic T-cellprecursors. Nat Biotechnol, 2008.    26(4): p. 453-61.-   17. Mardiros, A., et al., T cells expressing CD123-specific chimeric    antigen receptors exhibit specific cytolytic effector functions and    antitumor effects against human acute myeloid leukemia. Blood, 2013.    122(18): p. 3138-48.-   18. Pardoll, D. M., The blockade of immune checkpoints in cancer    immunotherapy. Nat Rev Cancer, 2012. 12(4): p. 252-64.-   19. Ascierto, P. A., et al., Clinical experiences with anti-CD137    and anti-PD-1 therapeutic antibodies. Semin Oncol, 2010. 37(5): p.    508-16.-   20. Blank, C., T. F. Gajewski, and A. Mackensen, Interaction of    PD-L1 on tumor cells with PD-1 on tumor-specific T cells as a    mechanism of immune evasion: implications for tumor immunotherapy.    Cancer Immunol Immunother, 2005. 54(4): p. 307-14.-   21. Keir, M. E., et al., PD-1 and its ligands in tolerance and    immunity. Annu Rev Immunol, 2008. 26: p. 677-704.-   22. Berger, R., et al., Phase I safety and pharmacokinetic study of    CT-011, a humanized antibody interacting with PD-1, in patients with    advanced hematologic malignancies. Clin Cancer Res, 2008. 14(10): p.    3044-51.-   23. Topalian, S. L., et al., Safety, activity, and immune correlates    ofanti-PD-1 antibody in cancer. N Engl J Med, 2012. 366(26): p.    2443-54.-   24. Brahmer, J. R., et al., Safety and activity of anti-PD-L1    antibody inpatients with advanced cancer. N Engl J Med, 2012.    366(26): p. 2455-65.-   25. Berthon, C., et al., In acute myeloid leukemia, B7-H1 (PD-L1)    protection of blasts from cytotoxic T cells is induced by TLR    ligands and interferon-gamma and can be reversed using MEK    inhibitors. Cancer Immunol Immunother, 2010. 59(12): p. 1839-49.

INFORMAL SEQUENCE LISTING (CDR L1): SEQ ID NO: 1 QSLLHSNGYKY (CDR L2):SEQ ID NO: 2 LGS (CDR L3): SEQ ID NO: 3 MQALQTPLT (CDR H1): SEQ ID NO: 4GYSFSSHW (CDR H2): SEQ ID NO: 5 IYPGDSDT (CDR H3): SEQ ID NO: 6ARGELPGEAYYFDN (Light chain variable region): SEQ ID NO: 7EIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYKYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTP LTFGGGTKVEIK(Heavy chain variable region): SEQ ID NO: 8QVQLVQSGAEVKKPGESLKISCKGSGYSFSSHWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARGE LPGEAYYFDNWGQGTLVTVSS(FR L1): SEQ ID NO: 9 EIVMTQSPLSLPVTPGEPASISCRSS (FR L2): SEQ ID NO: 10LDWYLQKPGQSPQLLIY (FR L3): SEQ ID NO: 11NRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC (FR L4): SEQ ID NO: 12 FGGGTKVEIK(FR H1): SEQ ID NO: 13 QVQLVQSGAEVKKPGESLKISCKGS (FR H2): SEQ ID NO: 14IGWVRQMPGKGLEWMGI (FRH3): SEQ ID NO: 15RYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYC (FR H4): SEQ ID NO: 16WGQGTLVTVSS (Amino acid sequence of 1D5-scFv): SEQ ID NO: 17QVQLVQSGAEVKKPGESLKISCKGSGYSFSSHWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARGELPGEAYYFDNWGQGTLVTVSSGGGGSGGGGSGGGGSEIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYKYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTPLTFGGGTKVEIK(Nucleotide sequence of 1D5 scFv): SEQ ID NO: 18CAGGTGCAGCTGGTGCAGAGCGGCGCCGAAGTGAAGAAGCCCGGCGAGAGCCTGAAGATCAGCTGCAAGGGCAGCGGCTACAGCTTCAGCAGCCACTGGATCGGCTGGGTCAGGCAGATGCCCGGCAAAGGCCTGGAGTGGATGGGCATCATCTATCCCGGCGACAGCGACACCAGGTACTCCCCCAGCTTCCAGGGCCAGGTGACCATCTCCGCCGACAAGAGCATTAGCACCGCCTACCTGCAGTGGAGCAGCCTCAAAGCCAGCGACACCGCCATGTACTACTGCGCCAGAGGCGAACTGCCCGGAGAGGCCTACTACTTCGATAACTGGGGCCAGGGCACCCTGGTGACAGTGAGCAGCGGAGGAGGCGGCAGCGGCGGCGGCGGATCCGGAGGAGGCGGCTCCGAGATCGTGATGACCCAGAGCCCTCTGAGCCTGCCCGTGACACCTGGCGAACCTGCCAGCATCAGCTGCAGAAGCAGCCAGAGCCTGCTCCACTCCAACGGCTACAAATACCTGGACTGGTATCTCCAGAAGCCTGGCCAGAGCCCCCAGCTGCTCATCTACCTGGGCAGCAATAGGGCCAGCGGAGTGCCCGACAGGTTTAGCGGCTCCGGAAGCGGCACCGATTTCACCCTGAAAATCAGCAGAGTGGAGGCCGAGGACGTGGGCGTGTACTACTGCATGCAGGCTCTGCAGACACCCCTGACCTTCGGCGGAGGCACCAAGGTGGAGATCAAG (Linker region):SEQ ID NO: 19 GGGGSGGGGSGGGGS (1D5-scFv-Hole-FC-LA sequence):SEQ ID NO: 20 QVQLVQSGAEVKKPGESLKISCKGSGYSFSSHWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARGELPGEAYYFDNWGQGTLVTVSSGGGGSGGGGSGGGGSEIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYKYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTPLTFGGGTKVEIKCAPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (Anti-CD3-scFv-Knob-FC-LA sequence):SEQ ID NO: 21 EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVLCAPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (1D5-scFv-CAR amino acid sequence)SEQ ID NO: 90 MLLLVTSLLLCELPHPAFLLIPQVQLVQSGAEVKKPGESLKISCKGSGYSFSSHWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARGELPGEAYYFDNWGQGTLVTVSSGGGGSGGGGSGGGGSEIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYKYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTPLTFGGGTKVEIKVAAAAFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLST ATKDTYDALHMQALPPR

P EMBODIMENTS

P Embodiment 1. An anti-interleukin-1 receptor accessory protein(IL1RAP) antibody comprising a light chain variable domain and a heavychain variable domain,

wherein said light chain variable domain comprises:

a CDR L1 as set forth in SEQ ID NO:1, a CDR L2 as set forth in SEQ IDNO:2 and a CDR L3 as set forth in SEQ ID NO:3; and

wherein said heavy chain variable domain comprises:

a CDR H1 as set forth in SEQ ID NO:4, a CDR H2 as set forth in SEQ IDNO:5, and a CDR H3 as set forth in SEQ ID NO:6.

P Embodiment 2. The antibody of P embodiment 1, wherein said light chainvariable domain comprises the sequence of SEQ ID NO:7.

P Embodiment 3. The antibody of P embodiment 1 or 2, wherein said heavychain variable domain comprises the sequence of SEQ ID NO:8.

P Embodiment 4. The antibody of one of P embodiments 1-3, wherein saidlight chain variable domain comprises a FR L1 as set forth in SEQ IDNO:9, a FR L2 as set forth in SEQ ID NO:10, FR L3 as set forth in SEQ IDNO:11 and a FR L4 as set forth in SEQ ID NO:12.

P Embodiment 5. The antibody of one of P embodiments 1-4, wherein saidheavy chain variable domain comprises a FR H1 as set forth in SEQ IDNO:13, a FR H2 as set forth in SEQ ID NO:14, FR H3 as set forth in SEQID NO:15 and a FR H4 as set forth in SEQ ID NO:16.

P Embodiment 6. The antibody of one of P embodiments 1-5, wherein saidantibody is an IgG.

P Embodiment 7. The antibody of one of P embodiments 1-6, wherein saidantibody is an IgG1.

P Embodiment 8. The antibody of one of P embodiments 1-5, wherein saidantibody is a Fab′ fragment.

P Embodiment 9. The antibody of one of P embodiments 1-5, wherein saidantibody is a single chain antibody (scFv).

P Embodiment 10. The antibody of one of P embodiments 1-5, wherein saidlight chain variable domain and said heavy chain variable domain formpart of a scFv.

P Embodiment 11. The antibody of P embodiment 9 or 10, wherein said scFvcomprises the sequence of SEQ ID NO:17.

P Embodiment 12. The antibody of one of P embodiments 1-11, wherein saidantibody is capable of binding IL1RAP with an equilibrium dissociationconstant (K_(D)) of about 21 nM.

P Embodiment 13. The antibody of one of P embodiments 1-11, wherein saidantibody has an EC₅₀ of about 4.9 ng/ml.

P Embodiment 14. The antibody of one of P embodiments 1-13, wherein saidantibody is bound to an IL1RAP.

P Embodiment 15. The antibody of P embodiment 14, wherein said IL1RAP isa human IL1RAP.

P Embodiment 16. The antibody of P embodiment 14 or 15, wherein saidIL1RAP forms part of a cell.

P Embodiment 17. The antibody of P embodiment 16, wherein said IL1RAP isexpressed on the surface of said cell.

P Embodiment 18. The antibody of P embodiment 16 or 17, wherein saidcell is a cancer cell.

P Embodiment 19. The antibody of P embodiment 18, wherein said cancercell is a leukemia stem cell (LSC).

P Embodiment 20. The antibody of P embodiment 18, wherein said cancercell is an acute myeloid leukemia (AML) cell.

P Embodiment 21. An isolated nucleic acid encoding an antibody of one ofP embodiments 1-21.

P Embodiment 22. A pharmaceutical composition comprising atherapeutically effective amount of an antibody of one of P embodiments1-21 and a pharmaceutically acceptable excipient.

P Embodiment 23. A method of treating cancer in a subject in needthereof, said method comprising administering to a subject atherapeutically effective amount of an antibody of one of P embodiments1-21, thereby treating cancer in said subject.

P Embodiment 24. A recombinant protein comprising:

(i) an antibody region comprising:

-   -   (a) a light chain variable domain comprising a CDR L1 as set        forth in SEQ ID NO:1, a CDR L2 as set forth in SEQ ID NO:2 and a        CDR L3 as set forth in SEQ ID NO:3; and    -   (b) a heavy chain variable region domain a CDR H1 as set forth        in SEQ ID NO:4, a CDR H2 as set forth in SEQ ID NO:5, and a CDR        H3 as set forth in SEQ ID NO:6; and

(ii) a transmembrane domain.

P Embodiment 25. The recombinant protein of P embodiment 24, whereinsaid light chain variable domain comprises the sequence of SEQ ID NO:7.

P Embodiment 26. The recombinant protein of P embodiment 24 or 25,wherein said heavy chain variable domain comprises the sequence of SEQID NO:8.

P Embodiment 27. The recombinant protein of one of P embodiments 24-26,wherein said light chain variable domain comprises a FR L1 as set forthin SEQ ID NO:9, a FR L2 as set forth in SEQ ID NO:10, FR L3 as set forthin SEQ ID NO:11 and a FR L4 as set forth in SEQ ID NO:12.

P Embodiment 28. The recombinant protein of one of P embodiments 24-27,wherein said heavy chain variable domain comprises a FR H1 as set forthin SEQ ID NO:13, a FR H2 as set forth in SEQ ID NO:14, FR H3 as setforth in SEQ ID NO:15 and a FR H4 as set forth in SEQ ID NO:16.

P Embodiment 29. The recombinant protein of one of P embodiments 24-28,wherein said antibody region comprises a single-chain variable fragment(scFv).

P Embodiment 30. The recombinant protein of P embodiment 29, whereinsaid scFv comprises the sequence of SEQ ID NO:17.

P Embodiment 31. The recombinant protein of one of P embodiments 24-30,wherein said recombinant protein is capable of binding IL1RAP with anequilibrium dissociation constant (K_(D)) of about 21 nM.

P Embodiment 32. The recombinant protein of one of P embodiments 24-31,wherein said recombinant protein has an EC₅₀ of about 4.9 ng/ml.

P Embodiment 33. The recombinant protein of one of P embodiments 24-32,wherein said recombinant protein is bound to an IL1RAP.

P Embodiment 34. The recombinant protein of P embodiment 33, whereinsaid IL1RAP is a human IL1RAP.

P Embodiment 35. The recombinant protein of P embodiment 33 or 34,wherein said IL1RAP forms part of a cell.

P Embodiment 36. The recombinant protein of one of P embodiments 33-35,wherein said IL1RAP is expressed on the surface of said cell.

P Embodiment 37. The recombinant protein of P embodiment 36, whereinsaid cell is a cancer cell.

P Embodiment 38. The recombinant protein P embodiment 37, wherein saidcancer cell is a leukemia stem cell (LSC).

P Embodiment 39. The recombinant protein P embodiment 37, wherein saidcancer cell is an acute myeloid leukemia (AML) cell.

P Embodiment 40. The recombinant protein of one of P embodiments 24-39,wherein said antibody region comprises an Fc domain.

P Embodiment 41. The recombinant protein of P embodiment 40, whereinsaid Fc domain is an IgG4 Fc domain.

P Embodiment 42. The recombinant protein of one of P embodiments 24-41,further comprising an intracellular co-stimulatory signaling domain.

P Embodiment 43. The recombinant protein of P embodiment 42, whereinsaid intracellular co-stimulatory signaling domain is a CD28intracellular co-stimulatory signaling domain, a 4-1BB intracellularco-stimulatory signaling domain, a ICOS intracellular co-stimulatorysignaling domain, or an OX-40 intracellular co-stimulatory signalingdomain.

P Embodiment 44. The recombinant protein of one of P embodiments 24-43,further comprising an intracellular T-cell signaling domain.

P Embodiment 45. The recombinant protein of P embodiment 44, whereinsaid intracellular T-cell signaling domain is a CD3 (intracellularT-cell signaling domain.

P Embodiment 46. The recombinant protein of one of P embodiments 24-45,further comprising a self-cleaving peptidyl sequence.

P Embodiment 47. The recombinant protein of P embodiment 46, whereinsaid self-cleaving peptidyl linker sequence is a T2A sequence or a 2Asequence.

P Embodiment 48. The recombinant protein of one of P embodiments 24-47,further comprising a detectable domain.

P Embodiment 49. The recombinant protein of P embodiment 48, whereinsaid detectable domain is a truncated EGFR (EGFRt) domain.

P Embodiment 50. The recombinant protein of one of P embodiments 24-49,wherein said recombinant protein forms part of a cell.

P Embodiment 51. The recombinant protein of one of P embodiments 24-49,wherein said recombinant protein forms part of a T cell.

P Embodiment 52. An isolated nucleic acid encoding a recombinant proteinof one of P embodiments 24-51.

P Embodiment 53. A pharmaceutical composition comprising atherapeutically effective amount of a recombinant protein of one of Pembodiments 24-51 and a pharmaceutically acceptable excipient.

P Embodiment 54. A method of treating cancer in a subject in needthereof, said method comprising administering to a subject atherapeutically effective amount of a recombinant protein of one of Pembodiments 24-51, thereby treating cancer in said subject.

P Embodiment 55. A recombinant protein comprising:

(i) a first antibody region capable of binding an effector cell ligand;and

(ii) a second antibody region, comprising:

-   -   (a) a light chain variable domain comprising a CDR L1 as set        forth in SEQ ID NO:1, a CDR L2 as set forth in SEQ ID NO:2 and a        CDR L3 as set forth in SEQ ID NO:3; and    -   (b) a heavy chain variable region domain a CDR H1 as set forth        in SEQ ID NO:4, a CDR H2 as set forth in SEQ ID NO:5, and a CDR        H3 as set forth in SEQ ID NO:6.

P Embodiment 56. The recombinant protein of P embodiment 55, whereinsaid effector cell ligand is a CD3 protein.

P Embodiment 57. The recombinant protein of one of P embodiments 55-56,wherein said light chain variable domain comprises the sequence of SEQID NO:7.

P Embodiment 58. The recombinant protein of one of P embodiments 55-57,wherein said heavy chain variable domain comprises the sequence of SEQID NO:8.

P Embodiment 59. The recombinant protein of one of P embodiments 55-58,wherein said light chain variable domain comprises a FR L1 as set forthin SEQ ID NO:9, a FR L2 as set forth in SEQ ID NO:10, FR L3 as set forthin SEQ ID NO:11 and a FR L4 as set forth in SEQ ID NO:12.

P Embodiment 60. The recombinant protein of one of P embodiments 55-59,wherein said heavy chain variable domain comprises a FR H1 as set forthin SEQ ID NO:13, a FR H2 as set forth in SEQ ID NO:14, FR H3 as setforth in SEQ ID NO:15 and a FR H4 as set forth in SEQ ID NO:16.

P Embodiment 61. The recombinant protein of one of P embodiments 55-60,wherein said first antibody region is a first Fab′ fragment or saidsecond antibody region is a second Fab′ fragment.

P Embodiment 62. The recombinant protein of one of P embodiments 55-60,wherein said first antibody region is a single chain variable fragment(scFv) or said second antibody region is a second single chain variablefragment (scFv).

P Embodiment 63. The recombinant protein of one of P embodiments 55-56,wherein said second scFv comprises the sequence of SEQ ID NO:17.

P Embodiment 64. The recombinant protein of one of P embodiments 55-63,wherein said second antibody region is capable of binding IL1RAP with anequilibrium dissociation constant (K_(D)) of about 21 nM.

P Embodiment 65. The recombinant protein of one of P embodiments 55-64,wherein said second antibody region has an EC₅₀ of about 4.9 ng/ml.

P Embodiment 66. The recombinant protein of one of P embodiments 55-65,wherein said second antibody region is bound to an IL1RAP.

P Embodiment 67. The recombinant protein of P embodiment 66, whereinsaid IL1RAP is a human IL1RAP.

P Embodiment 68. The recombinant protein of one of P embodiments 66-67,wherein said IL1RAP forms part of a cell.

P Embodiment 69. The recombinant protein of one of P embodiments 66-68,wherein said IL1RAP is expressed on the surface of said cell.

P Embodiment 70. The recombinant protein of P embodiment 69, whereinsaid cell is a cancer cell.

P Embodiment 71. The recombinant protein of P embodiment 70, whereinsaid cancer cell is a leukemia stem cell (LSC).

P Embodiment 72. The recombinant protein of P embodiment 70, whereinsaid cancer cell is an acute myeloid leukemia (AML) cell.

P Embodiment 73. A pharmaceutical composition comprising atherapeutically effective amount of a recombinant protein of one of Pembodiments 55-72 and a pharmaceutically acceptable excipient.

P Embodiment 74. A method of treating cancer in a subject in needthereof, said method comprising administering to a subject atherapeutically effective amount of a recombinant protein of one of Pembodiments 55-72, thereby treating cancer in said subject.

P Embodiment 75. The method of one of P embodiments 23, 54 or 74,wherein said cancer is leukemia.

P Embodiment 76. The method of one of P embodiments 23, 54 or 74,wherein said cancer is acute myeloid leukemia.

P Embodiment 77. The method of one of P embodiments 23, 54 or 74, saidmethod further comprising administering to said subject a secondtherapeutic agent.

P Embodiment 78. A method of inhibiting proliferation of a cell, saidmethod comprising:

(i) contacting a cell with an anti-IL1RAP antibody of one of Pembodiments 1-21, a recombinant protein of one of P embodiments 24-51 ora recombinant protein of one of P embodiments 55-72, thereby forming acontacted cell; and

(ii) allowing said anti-IL1RAP antibody of one of P embodiments 1-21,said recombinant protein of one of P embodiments 24-51 or saidrecombinant protein of one of P embodiments 55-72 to bind an IL1RAP onsaid contacted cell, thereby inhibiting proliferation of said cell.

P Embodiment 79. The method of P embodiment 78, wherein said cell is acancer cell.

P Embodiment 80. The method of P embodiment 78 or 79, wherein said cellis a leukemia stem cell (LSC).

1.-80. (canceled)
 81. An anti-interleukin-1 receptor accessory protein(IL1RAP) antibody comprising a light chain variable domain comprisingthe sequence of SEQ ID NO:7 and a heavy chain variable domain comprisingthe sequence of SEQ ID NO:8.
 82. The anti-IL1RAP antibody of claim 81,wherein said antibody is a Fab′ fragment.
 83. The anti-IL1RAP antibodyof claim 81, wherein said antibody is capable of binding IL1RAP with anequilibrium dissociation constant (K_(D)) of about 21 nM.
 84. Theanti-IL1RAP antibody of claim 81, wherein said antibody has an EC₅₀ ofabout 4.9 ng/ml.
 85. The anti-IL1RAP antibody of claim 81, wherein saidantibody is bound to an IL1RAP.
 86. An isolated nucleic acid encodingthe anti-IL1RAP antibody of claim
 81. 87. A method of treating cancer ina subject in need thereof, said method comprising administering to asubject a therapeutically effective amount of the anti-IL1RAP antibodyof claim 81, thereby treating cancer in said subject.
 88. Ananti-interleukin-1 receptor accessory protein (IL1RAP) single chainantibody (scFv) comprising a light chain variable domain comprising thesequence of SEQ ID NO:7 and a heavy chain variable domain comprising thesequence of SEQ ID NO:8.
 89. The anti-IL1RAP scFv of claim 88, whereinsaid scFv comprises the sequence of SEQ ID NO:17.
 90. An isolatednucleic acid encoding the anti-IL1RAP scFv of claim
 88. 91. Apharmaceutical composition comprising a therapeutically effective amountof the anti-IL1RAP scFv of claim 88 and a pharmaceutically acceptableexcipient.
 92. A method of treating cancer in a subject in need thereof,said method comprising administering to a subject a therapeuticallyeffective amount of the anti-IL1RAP scFv of claim 88, thereby treatingcancer in said subject.
 93. A recombinant protein comprising: (i) afirst single chain variable fragment (scFv) capable of binding aneffector cell ligand; and (ii) a second single chain variable fragment(scFv) capable of binding IL1RAP and comprising a light chain variabledomain comprising the sequence of SEQ ID NO:7 and a heavy chain variabledomain comprising the sequence of SEQ ID NO:8.
 94. The recombinantprotein of claim 93, wherein said effector cell ligand is a CD3 protein.95. The recombinant protein of claim 94, wherein said second scFvcomprises the sequence of SEQ ID NO:17.
 96. An isolated nucleic acidencoding the recombinant protein of claim
 93. 97. A pharmaceuticalcomposition comprising a therapeutically effective amount of therecombinant protein of claim 93 and a pharmaceutically acceptableexcipient.
 98. A method of treating cancer in a subject in need thereof,said method comprising administering to a subject a therapeuticallyeffective amount of the recombinant protein of claim 93, therebytreating cancer in said subject.
 99. A recombinant protein comprising:(i) an anti-IL1RAP single chain variable fragment (scFv) comprising alight chain variable domain comprising the sequence of SEQ ID NO:7 and aheavy chain variable domain comprising the sequence of SEQ ID NO:8; and(ii) a transmembrane domain.
 100. The recombinant protein of claim 99,wherein said anti-IL1RAP scFv comprises the sequence of SEQ ID NO:17.101. An isolated nucleic acid encoding the recombinant protein of claim99.
 102. A pharmaceutical composition comprising a therapeuticallyeffective amount of the recombinant protein of claim 99 and apharmaceutically acceptable excipient.
 103. A method of treating cancerin a subject in need thereof, said method comprising administering to asubject a therapeutically effective amount of the recombinant protein ofclaim 99, thereby treating cancer in said subject.
 104. A method ofinhibiting proliferation of a cell, said method comprising: (i)contacting a cell with an anti-IL1RAP antibody of claim 81, arecombinant protein of claim 93 or a recombinant protein of claim 99,thereby forming a contacted cell; and (ii) allowing said anti-IL1RAPantibody of claim 81, said recombinant protein of claim 93 or saidrecombinant protein of claim 99 to bind an IL1RAP on said contactedcell, thereby inhibiting proliferation of said cell.